Methods and compositions employing immunogenic fusion proteins

ABSTRACT

Compositions and methods are provided for the prevention and treatment of bacterial infections, including pneumococcal infections. Compositions provided herein comprise a variety of immunogenic fusion proteins, wherein at least one polypeptide component of a given fusion protein comprises a CbpA polypeptide and/or a cytolysoid polypeptide, or an active variant or fragment thereof. Methods are provided for the prevention and treatment of bacterial infections, including pneumococcal infections by employing the various immunogenic fusion proteins having at least one polypeptide component comprising a CbpA polypeptide and/or a cytolysoid polypeptide, or an active variant or fragment thereof.

FIELD OF THE INVENTION

The present invention relates to the field of vaccines for preventing ortreating pneumococcal infection.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named416012SEQLIST.txt, created on Feb. 21, 2012, and having a size of 82 KBand is filed concurrently with the specification. The sequence listingcontained in this ASCII formatted document is part of the specificationand is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae is a gram positive bacterium which is a majorcause of invasive infections such as sepsis, meningitis, otitis mediaand lobar pneumonia (Tuomanen et al. NEJM 322:1280-1284, 1995).Infection by S. pneumoniae remains a significant health threatworldwide. Pneumococci bind avidly to cells of the upper and lowerrespiratory tract and to endothelial cells present in blood vessels.Like most bacteria, adherence of pneumococci to human cells is achievedby presentation of bacterial surface proteins that bind to eukaryoticcell surface proteins (Cundell, D. & Tuomanen, E. (1994) Microb Pathog17:361-374). For example, bacteria translocate across cells of the upperrespiratory tract and nasopharynx via the polymeric immunoglobulinreceptor (pTgR) (Zhang et al. (2000) Cell 102:827-837). Alternatively,when the bacteria are in the blood stream, the pneumococcal bacteriabind to endothelial cells, and the bacteria cross the blood vesselendothelium and enter tissues by binding to and transcytosing with theplatelet activating factor (PAF) receptor (Cundell et al. (1995) Nature,377:435-438).

Current vaccines against S. pneumoniae employ purified carbohydrates ofthe capsules of up to the 23 most common serotypes of this bacterium,but such vaccines are only 50% protective against pneumonia (Shapiro etal. NJEM 325:1453, 1991) and are not immunogenic under the age of 2.Conjugate vaccines are based on pneumococcal capsular carbohydrateslinked to proteins such as diphtheria toxoid or tetanus toxoid.Protection against pneumonia, sepsis, or meningitis for these vaccinesis limited to the serotypes present in the formulation, thereby leavingpatients unprotected against most of the ninety-two serotypes of thisbacterium. Further, vaccines that are protective against both thecolonization of pneumococcal bacteria in the nasopharynx, as well, asagainst entry of pneumococcal bacteria into the bloodstream are neededin the art. Therefore, compositions and methods provided herein fills along felt need by providing pharmaceutical compositions (e.g., vaccines)for the prevention and treatment of a wide range of serotypes ofpneumococcal infections across all age groups.

BRIEF SUMMARY OF THE INVENTION

Compositions and methods are provided for the prevention and treatmentof bacterial infections, including pneumococcal infections. Compositionsprovided herein comprise a variety of immunogenic fusion proteins,wherein at least one polypeptide component of a given fusion proteincomprises a CbpA polypeptide and/or a cytolysoid polypeptide, or anactive variant or fragment thereof. Methods are provided for theprevention and treatment of bacterial infections, including pneumococcalinfections by employing the various immunogenic fusion proteins havingat least one polypeptide component comprising a CbpA polypeptide and/ora cytolysoid polypeptide, or an active variant or fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the domain structure of CbpA and the fragmentspresented herein.

FIG. 2 illustrates the tertiary structure of CbpA R2 domain and pointsout the R2₁ and R2₂ regions.

FIG. 3 demonstrates that the fusion protein elicits antibodies againstboth PdB and CbpA.

FIG. 4 demonstrates that when mice were challenged the following weekintranasally with 2.7×10⁷ S. pneumoniae T4X and followed for survival,the fusion protein elicited protection.

FIG. 5 provides the antibody titers for various fusion proteins.

FIG. 6 provides the percent protection against meningitis for variousfusion proteins.

FIG. 7 provides the percent survival for various fusion proteins.

FIG. 8 provides antibody titers for various fusion proteins.

FIG. 9 provides the percent survival for various fusion proteins.

FIG. 10 provides antibody titers for various fusion proteins.

FIG. 11 shows the percent survival following administration of variousfusion proteins.

FIG. 12 shows the antibody titers of various fusion proteins.

FIG. 13 shows the use of an anti-sera against L460D, YPT-L460D-NEEK andPBS (−) control in an ELISA based assay for recognition of S. pneumoniaeT4R whole bacteria.

FIG. 14 shows the percent survival of various fusion proteins.

FIG. 15 shows antibody titers for various fusion proteins.

FIG. 16 shows the percent survival of the mice after challenge withvarious fusion proteins.

FIG. 17 shows the percent survival upon administration of various fusionproteins.

FIG. 18 shows that the fusion protein is protective against meningitiscompared to toxoid alone or negative control.

FIG. 19 shows that various fusion toxoid showed enhanced survival forbacterial challenges.

FIG. 20 shows the number of positive spots per well reactive to CbpA orpneumolysis.

FIG. 21 summarizes the percent survival and percent of meningitis.

FIG. 22 provides data showing the frequency and time to development ofmeningitis.

FIG. 23 provides data showing the survival time for mice immunized withL460D, YPT-L460D-NEEK or Alum (−) control.

FIG. 24 shows mice immunized with YPT-L460D-NEEK or L460D were protectedsimilarly and both were significantly better than adjuvant alone.

FIG. 25 L460D and YPT-L460D-NEEK show best protection followed byL460D-NEEK.

FIG. 26 shows data demonstrating toxoid constructs remainednonhemolytic.

FIG. 27 shows pneumolysin neutralization assays using antisera fromimmunized mice.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

Compositions and methods are provided for the prevention and treatmentof bacterial infections, including pneumococcal infections. Compositionsprovided herein comprise immunogenic fusion proteins comprising multipleoperably linked polypeptides, wherein at least one polypeptide componentof a given fusion protein comprises a CbpA polypeptide and/or acytolysoid polypeptide, or an active variant or fragment thereof. Thefusion of multiple genes encoding polypeptides together resulting in onefusion protein minimizes cost of vaccine production, and continues toallow for designs which provide efficacy to a breadth of serotypes.

The fusion proteins disclosed herein are immunogenic. As used herein, an“immunogen” is a substance that induces an immune response. The term“immunogenic” refers to the ability of a substance to induce an immuneresponse when administered to an animal. A substance such as apolypeptide displays “increased immunogenicity” relative to anotherpolypeptide when administration of the first polypeptide to an animalresults in a greater immune response than that observed withadministration of the other polypeptide. An increase in immunogenicitycan also refer to not only a greater response in terms of the productionof more antibody or T cells but also the production of more protectiveantibody or T cells. Thus, in specific embodiments, an increase inimmunogenicity refers to any statistically significant increase in thelevel of antibodies or T cells or antibody or T cell production or anystatistically significant increase in a protective antibody response.Such an increase can include a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100% or higher increase in the level of antibodies or in theprotective antibody response. The immunogenicity of a polypeptide can beassayed for by measuring the level of antibodies or T cells producedagainst the polypeptide. Assays to measure for the level of antibodiesare known, for example, see Harlow and Lane (1988) Antibodies, ALaboratory Manual (Cold Spring Harbor Publications, New York), for astandard description of antibody generation, immunoassay formats andconditions that can be used to determine specific immunoreactivity.Assays for T cells specific to a polypeptide are known, as shown inExample 7 herein, or see, for example, Rudraraju et al. (2011) Virology410:429-36, herein incorporated by reference. In other instances,increased immunogenicity can be detected as an improved clinicaloutcome, as discussed elsewhere herein.

I. Compositions

Compositions disclosed herein provide fusion proteins comprising a firstpolypeptide operably linked to a second polypeptide. As used herein,“fusion protein” refers to the in frame genetic linkage of at least twoheterologous polypeptides. Upon transcription/translation, a singleprotein is made. In this way, multiple proteins, or fragments thereofcan be incorporated into a single polypeptide. “Operably linked” isintended to mean a functional linkage between two or more elements. Forexample, an operable linkage between two polypeptides fuses bothpolypeptides together in frame to produce a single polypeptide fusionprotein. In particular aspects, the fusion protein further comprises athird polypeptide. Multiple forms of immunogenic fusion proteins aredisclosed herein and discussed in detail below.

A. Fusion Proteins Comprising Immunogenic Regions of Choline BindingProtein A (CbpA)

i. CbpA

Compositions and methods are provided comprising immunogenic fusionproteins comprising immunogenic regions of the Choline Binding Protein A(CbpA). As used herein, a “CbpA fusion protein” can comprise the fullCbpA polypeptide or active variants or fragments thereof or anyimmunogenic fragment of CbpA as discussed in further detail elsewhereherein. CbpA is a 75 kD surface-exposed choline binding protein ofStreptococcus pneumoniae. CbpA binds several ligands in the hostincluding pIgR, C3, factor H and laminin receptor. The N-terminus ofCbpA (region without the terminal choline binding domain) containsnumerous repeats of the leucine zipper motif that cluster within 5domains termed the A, B, R1, R2, and C domains (FIG. 1). The R2 domainof CbpA (amino acid residues approximately 329 to 443) comprises threeanti-parallel alpha-helices (FIG. 2). This three alpha-helix structureis similarly predicted for the R1 domain (Jordan et al. (2006) J. Am.Chem. Soc. 128(28):9119-9128). Notably, the R domains from the Tigr4strain of S. pneumoniae are highly conserved among CbpA sequences fromother pneumococcal strains.

While any immunogenic fragment or domain of CbpA can be used in thefusion proteins disclosed herein, in one embodiment, the fusion proteincomprises at least one R2 domain or active variant or fragment of the R2domain. The R2 domain of CpbA comprises two regions, R2₁ and R2₂, whichhave been shown to form a loop conformation at each of the two turns ofthe anti-parallel alpha-helices in the three-dimensional structure ofthe R2 domain. See FIG. 2 boxes. As discussed in U.S. Patent PublicationNo. 2010-0143394-A1, herein incorporated by reference, the loopconformation of the R2₁ and R2₂ regions increases the immunogenicity ofthe R2 regions. Thus, the fusion proteins disclosed herein can compriseat least one immunogenic fragment or variant of the R2 domain of CbpA,such as, as least 1, 2, 3, 4, or more copies of the R2 domain, the R2₁region and/or the R2₂ region or active variants and fragments thereof.

The R2₁ and R2₂ regions of CbpA have defined functions in disease. TheR2₁ region comprises the pIgR binding site. Binding of the R2₁ region ofCbpA to the pIgR allows the pneumococcal bacteria to utilize endocytosismachinery to translocate across nasopharyngeal epithelial cells into theblood stream. This step is critical for bacterial colonization of thenasopharynx and entry of the bacteria into the blood stream.

The R2₁ polypeptide comprises the amino acid sequence set forth in SEQID NO: 1 or active variants or fragments thereof. In some embodiments,the immunogenic fusion proteins comprising at least one copy of the R2₁region or active variants and fragments thereof can produce animmunogenic response which targets bacterial pIgR binding andcolonization of the nasopharynx and entry into the blood stream.

The R2₂ region of CbpA comprises the laminin receptor binding site. Whenthe R2₂ region of CbpA binds to the laminin receptor, it facilitates thehand off of the bacterium to platelet activating factor (PAF) receptorwhich carries the bacterium into the endothelial cell, across the bloodvessel wall, out of the blood stream and into the tissues. Binding tothe laminin receptor is a critical step for bacteria to cross the bloodbrain barrier and cause meningitis. The R2₂ polypeptide comprises theamino acid sequence set forth in SEQ ID NO: 2 or active variants orfragments thereof. In some embodiments, the immunogenic fusion proteinscomprising the R2₂ region of CbpA or active variants and fragmentsthereof can produce an immunogenic response which targets lamininreceptor binding, and thus the ability of the bacteria to cross theblood brain barrier and cause meningitis.

In light of the different activities of the R2₁ and R2₂ regions of CbpA,the immunogenic fusion proteins described herein can comprise one ormore copies of the R2 regions or an active variant or fragment thereof,one or more copies of either the R2₁ region or the R2₂ region or activevariants and fragment thereof, or a combination of both the R2₁ and R2₂regions or active variant and fragments thereof. In view of thedifferent functional aspects of the R2₁ and R2₂ regions, one can therebydesign a fusion protein having immunogenic activity.

In specific embodiments, the R2₁ and/or R2₂ polypeptide or activevariants and fragments thereof employed in the immunogenic fusionprotein comprises a loop conformation similar to that present in thenative protein. By “loop conformation” is intended a three dimensionalprotein structure stabilized in a loop structure by a synthetic linkagein the polypeptide. As used herein, a “synthetic linkage” comprises anycovalent or non-covalent interaction that is created in the polypeptidesthat does not occur in the native protein. Any form of a syntheticlinkage that can form a covalent or non-covalent bond between aminoacids in the native or variant polypeptides can be used. Such syntheticlinkages can include synthetic peptide bonds that are engineered tooccur between amino acids present in either the native polypeptide or avariant thereof. The R2₁ and R2₂ polypeptides or active variants andfragments thereof may comprise any form of synthetic linkage that canresult in the formation of a covalent bond between amino acids in thenative CbpA protein or variant thereof. A synthetic linkage furtherincludes any non-covalent interaction that does not occur in the nativepolypeptide. For example, loop polypeptides comprising the R2₁ and/orR2₂ region may be engineered to have cysteine residues that are notpresent in the native CbpA protein and that allow for the formation of adisulfide bridge that stabilizes the polypeptide in a loop conformation.Various methods are known in the art to form such loop conformations ina polypeptide. See, for example, Chhabra et al. (1998) Tetrahedron Lett.39:1603-1606; Rohwedder et al. (1998) Tetrahedron Lett. 39:1175-1178;Wittmann & Seeberger (2000) Angew. Chem. Int. Ed. Engl. 39:4348-4352;and Chan et al. (1995) J. Chem. Soc., Chem. Commun. 21:2209-2210, all ofwhich are herein incorporated by reference in their entirety.Non-limiting examples of R2₁ or R2₂ polypeptides with a loopconformation are discussed in, for example, U.S. Patent Publication No.2010-0143394-A1, which is herein incorporated by reference in itsentirety.

In one embodiment, the loop conformation of the R2₁ and R2₂ polypeptidesis generated by at least a first cysteine residue and a second cysteineresidue, where the first and the second cysteine residues form adisulfide bond such that the polypeptide is stabilized in a loopconformation. In some specific embodiments, the cysteine residues can beadded to the N-terminal and C-terminal ends of the R2₁ and R2₂polypeptides, or the cysteine residues may be added internally bysubstituting amino acids within the polypeptide sequence with cysteineresidues such that the R2₁ and R2₂ polypeptides form a loopconformation. While not intending to be limited to a particularmechanism, it is believed that stabilization of the R2₁ and R2₂polypeptides in a loop conformation more closely mimics the nativeconformation of these polypeptides within the CbpA protein. The R2₁ andR2₂ loop polypeptides thereby have increased protective immunogenicityrelative to those polypeptides that are not stabilized in the loopconformation (e.g., linear versions of these polypeptides).

In one non-limiting embodiment, the looped R2₁ and R2₂ polypeptides oractive variant or fragments thereof employed in the immunogenic fusionproteins have cysteine substitutions as set forth in SEQ ID NOS: 3 or 4,or active variants or fragments thereof. SEQ ID NO: 3 (AKA YPT)comprises amino acid residues 329-391 of the CbpA protein, wherein thevaline at position 333 and the lysine at position 386 have each beensubstituted with a cysteine residue. SEQ ID NO: 4 (AKA NEEK) comprisesamino acid residues 361-443 of the CbpA protein, wherein the lysine atposition 364 and the valine at position 439 have each been substitutedwith a cysteine residue.

Active variants and fragments of the full-length CbpA polypeptide (SEQID NO: 12), the CbpA polypeptide without the choline binding domain(R1R2, SEQ ID NO: 13), the R2 domain of the CbpA polypeptide (SEQ ID NO:14), the R2₁ region (SEQ ID NOS: 1 or 3) and/or the R2₂ region (SEQ IDNOS: 2 or 4) can be employed in the various fusion proteins disclosedherein. Such active variants can comprise at least 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to SEQ ID NOS:1, 2, 3, 4, 12, 13 or 14, wherein the activevariants retain biological activity and hence are immunogenic.Non-limiting examples of R2₁ and R2₂ polypeptide variants are disclosed,for example, in U.S. Patent Publication No. 2010-0143394-A1, hereinincorporated by reference. Active fragment can comprises amino acidsequences having at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80,100, 150, or more consecutive amino acids of any one of SEQ ID NOS: 1,2, 3, 4, 12, 13 or 14, where the active fragments retain biologicalactivity and hence are immunogenic.

ii. Other Components of CbpA Fusion Proteins

The immunogenicity of the fusion proteins disclosed herein can beincreased through the addition of a heterologous T cell epitope (TCE).Thus, the fusion proteins disclosed herein further comprise at least oneheterologous TCE fused in frame to a bacterial polypeptide or variant orfragment thereof (i.e. the CbpA polypeptide or active variant andfragment thereof). Thus, for example, an amino acid sequence for a TCEmay be linked to a CbpA polypeptide or active variant or fragmentthereof to increase the immunogenicity of the polypeptide relative tothat of the same polypeptide lacking the TCE sequence.

As used herein, a “TCE” refers to a polypeptide sequence recognized by Tcells. See, for example, El Kasmi et al. (2000) J. Gen. Virol.81:729-735 and Obeid et al. (1995) J. Virol. 69:1420-1428; El Kasmi etal. (1999) Vaccine 17:2436-2445; El Kasmi et al. (1998) Mol. Immunol.35:905-918; El Kasmi et al. (2000) J. Gen. Virol. 81:729-735; Obeid etal. (1995) J. Virol. 69:1420-1428; and Bouche et al. (2005) Vaccine23:2074-2077. Polypeptides comprising a TCE sequence are generallybetween about 10-30, 30-50 or 50-90, or 90-100 amino acids, or up to afull length protein. While any amino acid sequence having a TCE can beused in the in the fusion proteins disclosed herein, non-limitingexamples of TCE sequences are set forth in SEQ ID NOS: 15 and 16, oractive variants and fragments thereof.

“Heterologous” in reference to a polypeptide is a polypeptide thatoriginates from a different protein. The heterologous TCE sequence canoriginate from the same organism as the other polypeptide component ofthe fusion protein, or the TCE can be from a different organism than theother polypeptide components of the fusion protein.

In a specific embodiment, an immunogenic CbpA fusion protein comprises afirst polypeptide having an R2₁ or R2₂ region of CbpA, for example, theamino acid sequence of SEQ ID NOS: 1, 2, 3 or 4, or active variants orfragments thereof, wherein the first polypeptide comprising either theR2₁ or R2₂ region of CbpA forms a loop conformation and is immunogenic,and the fusion protein comprises a second polypeptide comprising atleast one heterologous TCE, fused in frame to the first polypeptide.

In some embodiments, the heterologous TCE employed in the CbpA fusionprotein disclosed herein comprises an immunogenic pneumococcalpolypeptide or an active variant or fragment thereof. In suchembodiments, in addition to enhancing the immunogenicity of the firstpolypeptide by providing a TCE, employment of a second immunogenicpneumococcal polypeptide in the CbpA fusion proteins described hereinprovides another means to target the pneumococcal bacteria and improveimmunogenicity against pneumococcal infections. Non-limiting examples ofimmunogenic pneumococcal proteins which can be employed in the CbpAfusion proteins disclosed herein, include, pneumolysin, pneumococcalsurface protein A (PspA), neuraminidase A (nanA),β-N-acetylhexosaminidase (StrH), DnaK, or AliB protein or active variantand fragments thereof. Additional immunogenic pneumococcal polypeptidesare known in the art and can be found, for example, in U.S. Pat. No.6,042,838, U.S. Pat. No. 6,232,116, U.S. Patent Publication No.2009/0170162A1, C. C. Daniels et al. (2010) Infection and Immunity78:2163-72, and Zysk et al. (2000) Infection and Immunity 68:3740-3743,each of which is herein incorporated by reference in their entirety.

In one embodiment, the TCE of the CbpA fusion protein comprises apneumolysoid polypeptide or a variant or fragment thereof. Pneumolysinis a pore forming toxin and is the major cytolysin produced byStreptococcus pneumoniae. Pneumolysin oligomerizes to form pores in cellmembranes, and facilitates intrapulmonary bacterial growth and entryinto the blood stream by its hemolytic and complement activatingproperties. The amino acid sequence of wild-type or native pneumolysinis set forth in SEQ ID NO: 5. As used herein, “pneumolysoid” refers to amodified pneumolysin (a pneumolysin toxoid), wherein the modification ofthe protein inactivates or reduces the oligomerization, hemolytic and/orcomplement activating properties of the pneumolysoid protein while stillretaining immunogenic activity. A reduction in the toxicity of thepneumolysin protein (i.e. a reduction in oligomerization, hemolysis,and/or complement activation) comprises at least a 1%, 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% or greater statistically significantdecrease relative to an appropriate control. Various methods to assayfor pneumolysin activity are known in the art. For example, Example 10described elsewhere herein, provides a detailed assay to determinehemolytic activity of pneumolysoids. Complement activation may bedetermined, for example, by a two-dimensional gel electrophoresis assayto detect conversion of C3. See, J. C. Paton et al. (1984) Infection andImmunity 43:1085-1087, herein incorporated by reference. Oligomerizationof pneumolysin may be assessed, for example, by a combination of sucrosedensity gradient centrifugation and gel electrophoresis as described inF. D. Saunders et al. (1989) Infection and Immunity 57:2547-2552, hereinincorporated by reference. Various pneumolysoids that can be employed inthe various immunogenic fusion proteins provided herein are describedin, for example, WO2005/108419, WO2005/108580, WO 90/06951, U.S. PatentApplication No. 2009/0285846A1 and U.S. Patent Application No.2010/0166795, which are herein incorporated by reference. WO2005/108419and WO2005/108580 disclose pneumolysoids having a mutation (e.g. asubstitution or deletion) within the region of amino acids 144 to 161 ofthe wild-type pneumolysin protein. These mutants have reducedoligomerization and/or hemolytic activity as compared to the wild-typepneumolysin, and are therefore less toxic. The mutant may have asubstitution or deletion of one or more amino acids 144 to 161 of thewild-type pneumolysin sequence. Thus, the pneumolysoid may have amutation at one or more of the amino acid residues 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161of wild-type pneumolysin. In addition, pneumolysoids having reducedhemolytic activity and having at least one amino acid substitution ordeletion in at least one of the regions corresponding to amino acids257-297, 367-397 or 424-437 of the wild-type pneumolysin are describedin WO 90/06951.

The pneumolysoid set forth in SEQ ID NO: 7, or an active variant orfragment thereof, comprises a mutation of the lysine at amino acidposition 460 to an aspartic acid residue (L460D) which renders thepneumolysoid non-hemolytic. This pneumolysoid is referred to herein asthe “L460D” pneumolysoid and is disclosed in U.S. Patent Application No.2009/0285846A1, herein incorporated by reference in its entirety. Anactive variant of SEQ ID NO: 7 is provided herein and is set forth inSEQ ID NO: 39. The active variant comprises an amino acid change fromLysine at position 208 to Arginine when compared to SEQ ID NO: 7.

The pneumolysoid set forth in SEQ ID NO:8, or an active variant orfragment thereof, comprises a substitution of asparagine in place ofaspartic acid at amino acid position 385 and deletion of alanine 146 andarginine 147 of the wild-type pneumolysin sequence (Δ6N385pneumolysoid). This Δ6N385 pneumolysoid is deficient in both hemolysisand complement activation and is disclosed in U.S. Patent ApplicationNo. 2010/0166795 and in T. J. Mitchell et al. (1991) MolecularMicrobiology 5:1883-1888, herein incorporated by reference in theirentirety.

The pneumolysoid set forth in SEQ ID NO: 17, or an active variant orfragment thereof, comprises an amino acid substitution of phenylalaninein place of tryptophan at amino acid position 433 of the wild-typepneumolysin sequence (PdB). This PdB pneumolysoid is deficient inhemolysis and is disclosed in U.S. Pat. No. 6,716,432, hereinincorporated by reference in its entirety.

Active variants or fragments of the various pneumolysoids are providedherein. Such active variants can comprise at least 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to SEQ ID NOS: 5, 7, 8, 17 or 39. An active variant will retainimmunogenic activity. Active variants of pneumolysin are well known inthe art and find use as pneumolysoids. See, for example, US 2010/0166795and US 2009/0285846A1, herein incorporated by reference. The artprovides substantial guidance regarding the preparation of suchvariants, as described elsewhere herein. Thus, in one embodiment, theimmunogenic CbpA fusion proteins can comprise the pneumolysoid set forthin SEQ ID NO: 7, 8, 17 or 39 or an active variant thereof having atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%sequence identity to the amino acid sequence of SEQ ID NO: 7, 8, 17 or39, wherein the active variant is immunogenic.

iii. Non-Limiting Examples of CbpA/TCE Fusion Proteins

The immunogenic polypeptides as disclosed herein can be operably linkedin a variety of ways to produce an immunogenic fusion protein. When asingle CbpA polypeptide or active variant or fragment thereof isemployed, the TCE can be fused to the N-terminal end or the C-terminalend of the CbpA polypeptide or active variant or fragment thereof. Thefusion protein may comprise other protein components such as a linkerpeptide between the polypeptides of the fusion protein, or a peptide tagfor affinity purification (for example at the N- or C-terminus).

In other embodiments, the CbpA immunogenic fusion proteins can compriseat least 1, 2, 3, 4, 5 or more of the R2₁ or R2₂ regions, or activevariants or fragments thereof, operably linked to a heterologous TCE. Inone embodiment, the immunogenic fusion protein can comprise a thirdpolypeptide fused in frame to a first polypeptide or a secondpolypeptide comprising a TCE, wherein the third polypeptide is from abacteria and is immunogenic. When multiple CbpA polypeptides or variantsand fragments thereof are employed in the fusion protein, the TCE can befound at either the N-terminal or C-terminal end of the fusion protein,or alternatively can be located internally in the fusion protein so thatit is flanked by CbpA polypeptide sequences. Using multiple regions ofthe same protein in the fusion protein, in combination with a TCE, mayincrease immunogenicity to the protein by inducing antibody responses tomultiple regions of the protein.

In one embodiment, the immunogenic fusion protein comprises an R2₁ orR2₂ polypeptide in a loop conformation (i.e. SEQ ID NOS: 1, 2, 3 or 4)or active variants or fragments thereof, fused in frame to aheterologous TCE (i.e. a pneumococcal polypeptide or a pneumolysoidpolypeptide such as those in SEQ ID NOS: 5, 7, 8, 17 or 39) or activevariants or fragments thereof, fused in frame to a second R2₁ or R2₂polypeptide in a loop conformation (i.e. SEQ ID NOS: 1, 2, 3 or 4) oractive variants or fragments thereof. Table 1 provides a non-limitinglist of the various structures encompassed by the CbpA fusion proteinsdisclosed herein.

In a specific embodiment, the immunogenic CbpA fusion protein comprisesan R2₁ polypeptide comprising SEQ ID NOS: 1 or 3 or an active variant orfragment thereof in a loop conformation, the L460D pneumolysoid of SEQID NO: 7 or 39 or an active variant or fragment thereof, and an R2₂polypeptide comprising SEQ ID NOS: 2 or 4 or an active variant orfragment thereof in a loop conformation. In a particular embodiment theimmunogenic fusion protein comprises the amino acid sequence set forthin SEQ ID NO: 9 or an active variant or fragment thereof.

In other non limiting embodiments, the immunogenic fusion proteincomprises an R2₁ polypeptide comprising SEQ ID NOS: 1 or 3 or an activevariant or fragment thereof in a loop conformation, the Δ6N385pneumolysoid of SEQ ID NO: 8 or an active variant or fragment thereof,and an R2₂ polypeptide comprising SEQ ID NOS: 2 or 4 or an activevariant or fragment thereof in a loop conformation. In a particularembodiment the immunogenic fusion protein comprises the amino acidsequence set forth in SEQ ID NO: 11 or an active variant or fragmentthereof.

TABLE 1 Examples of CbpA fusion proteins* First Polypeptide SecondPolypeptide Third Polypeptide SEQ ID NOs: 1, 2, 3, 4 or active TCE Anybacterial polypeptide variants or fragments thereof SEQ ID NOs: 1, 2, 3,4 or active Pneumococcal polypeptide or variants or fragments thereofactive variant or fragment thereof SEQ ID NOs: 1, 2, 3, 4 or activePneumolysoid or active variant or variants or fragments thereof fragmentthereof SEQ ID NOs: 1, 2, 3, 4 or active L460D (SEQ ID NO: 7 or 39 orvariants or fragments thereof active variant or fragment thereof) SEQ IDNOs: 1, 2, 3, 4 or active Δ6N385 (SEQ ID NO: 8 or active variants orfragments thereof variant or fragment thereof) SEQ ID NOs: 1, 2, 3, 4 oractive PdB (SEQ ID NO: 17 or active variants or fragments thereofvariant or fragment thereof) SEQ ID NO: 1 or an active Pneumococcalpolypeptide or SEQ ID NO: 1 or an active variant or fragment thereofactive variant or fragment thereof variant or fragment thereof SEQ IDNO: 1 or an active Pneumolysoid or active variant or SEQ ID NO: 1 or anactive variant or fragment thereof fragment thereof variant or fragmentthereof SEQ ID NO: 1 or an active L460D (SEQ ID NO: 7 or 39 or SEQ IDNO: 1 or an active variant or fragment thereof active variant orfragment thereof) variant or fragment thereof SEQ ID NO: 1 or an activeΔ6N385 (SEQ ID NO: 8 or active SEQ ID NO: 1 or an active variant orfragment thereof variant or fragment thereof) variant or fragmentthereof SEQ ID NO: 1 or an active PdB (SEQ ID NO: 17 or active SEQ IDNO: 1 or an active variant or fragment thereof variant or fragmentthereof) variant or fragment thereof SEQ ID NO: 1 or an activePneumococcal polypeptide or SEQ ID NO: 2 or an active variant orfragment thereof active variant or fragment thereof variant or fragmentthereof SEQ ID NO: 1 or an active Pneumolysoid or active variant or SEQID NO: 2 or an active variant or fragment thereof fragment thereofvariant or fragment thereof SEQ ID NO: 1 or an active L460D (SEQ ID NO:7 or 39 or SEQ ID NO: 2 or an active variant or fragment thereof activevariant or fragment thereof) variant or fragment thereof SEQ ID NO: 1 oran active Δ6N385 (SEQ ID NO: 8 or active SEQ ID NO: 2 or an activevariant or fragment thereof variant or fragment thereof) variant orfragment thereof SEQ ID NO: 1 or an active PdB (SEQ ID NO: 17 or activeSEQ ID NO: 2 or an active variant or fragment thereof variant orfragment thereof) variant or fragment thereof SEQ ID NO: 1 or an activePneumococcal polypeptide or SEQ ID NO: 4 or an active variant orfragment thereof active variant or fragment thereof variant or fragmentthereof SEQ ID NO: 1 or an active Pneumolysoid or active variant or SEQID NO: 4 or an active variant or fragment thereof fragment thereofvariant or fragment thereof SEQ ID NO: 1 or an active L460D (SEQ ID NO:7 or 39 or SEQ ID NO: 4 or an active variant or fragment thereof activevariant or fragment thereof) variant or fragment thereof SEQ ID NO: 1 oran active Δ6N385 (SEQ ID NO: 8 or active SEQ ID NO: 4 or an activevariant or fragment thereof variant or fragment thereof) variant orfragment thereof SEQ ID NO: 1 or an active PdB (SEQ ID NO: 17 or activeSEQ ID NO: 4 or an active variant or fragment thereof variant orfragment thereof) variant or fragment thereof SEQ ID NO: 2 or an activePneumococcal polypeptide or SEQ ID NO: 1 or an active variant orfragment thereof active variant or fragment thereof variant or fragmentthereof SEQ ID NO: 2 or an active Pneumolysoid or active variant or SEQID NO: 1 or an active variant or fragment thereof fragment thereofvariant or fragment thereof SEQ ID NO: 2 or an active L460D (SEQ ID NO:7 or 39 or SEQ ID NO: 1 or an active variant or fragment thereof activevariant or fragment thereof) variant or fragment thereof SEQ ID NO: 2 oran active Δ6N385 (SEQ ID NO: 8 or active SEQ ID NO: 1 or an activevariant or fragment thereof variant or fragment thereof) variant orfragment thereof SEQ ID NO: 2 or an active PdB (SEQ ID NO: 17 or activeSEQ ID NO: 1 or an active variant or fragment thereof variant orfragment thereof) variant or fragment thereof SEQ ID NO: 2 or an activePneumococcal polypeptide or SEQ ID NO: 2 or an active variant orfragment thereof active variant or fragment thereof variant or fragmentthereof SEQ ID NO: 2 or an active Pneumolysoid or active variant or SEQID NO: 2 or an active variant or fragment thereof fragment thereofvariant or fragment thereof SEQ ID NO: 2 or an active L460D (SEQ ID NO:7 or 39 or SEQ ID NO: 2 or an active variant or fragment thereof activevariant or fragment thereof) variant or fragment thereof SEQ ID NO: 2 oran active Δ6N385 (SEQ ID NO: 8 or active SEQ ID NO: 2 or an activevariant or fragment thereof variant or fragment thereof) variant orfragment thereof SEQ ID NO: 2 or an active PdB (SEQ ID NO: 17 or activeSEQ ID NO: 2 or an active variant or fragment thereof variant orfragment thereof) variant or fragment thereof SEQ ID NO: 2 or an activePneumococcal polypeptide or SEQ ID NO: 3 or an active variant orfragment thereof active variant or fragment thereof variant or fragmentthereof SEQ ID NO: 2 or an active Pneumolysoid or active variant or SEQID NO: 3 or an active variant or fragment thereof fragment thereofvariant or fragment thereof SEQ ID NO: 2 or an active L460D (SEQ ID NO:7 or 39 or SEQ ID NO: 3 or an active variant or fragment thereof activevariant or fragment thereof) variant or fragment thereof SEQ ID NO: 2 oran active Δ6N385 (SEQ ID NO: 8 or active SEQ ID NO: 3 or an activevariant or fragment thereof variant or fragment thereof) variant orfragment thereof SEQ ID NO: 2 or an active PdB (SEQ ID NO: 17 or activeSEQ ID NO: 3 or an active variant or fragment thereof variant orfragment thereof) variant or fragment thereof SEQ ID NO: 3 or an activePneumococcal polypeptide or SEQ ID NO: 2 or an active variant orfragment thereof active variant or fragment thereof variant or fragmentthereof SEQ ID NO: 3 or an active Pneumolysoid or active variant or SEQID NO: 2 or an active variant or fragment thereof fragment thereofvariant or fragment thereof SEQ ID NO: 3 or an active L460D (SEQ ID NO:7 or 39 or SEQ ID NO: 2 or an active variant or fragment thereof activevariant or fragment thereof) variant or fragment thereof SEQ ID NO: 3 oran active Δ6N385 (SEQ ID NO: 8 or active SEQ ID NO: 2 or an activevariant or fragment thereof variant or fragment thereof) variant orfragment thereof SEQ ID NO: 3 or an active PdB (SEQ ID NO: 17 or activeSEQ ID NO: 2 or an active variant or fragment thereof variant orfragment thereof) variant or fragment thereof SEQ ID NO: 3 or an activePneumococcal polypeptide or SEQ ID NO: 3 or an active variant orfragment thereof active variant or fragment thereof variant or fragmentthereof SEQ ID NO: 3 or an active Pneumolysoid or active variant or SEQID NO: 3 or an active variant or fragment thereof fragment thereofvariant or fragment thereof SEQ ID NO: 3 or an active L460D (SEQ ID NO:7 or 39 or SEQ ID NO: 3 or an active variant or fragment thereof activevariant or fragment thereof) variant or fragment thereof SEQ ID NO: 3 oran active Δ6N385 (SEQ ID NO: 8 or active SEQ ID NO: 3 or an activevariant or fragment thereof variant or fragment thereof) variant orfragment thereof SEQ ID NO: 3 or an active PdB (SEQ ID NO: 17 or activeSEQ ID NO: 3 or an active variant or fragment thereof variant orfragment thereof) variant or fragment thereof SEQ ID NO: 3 or an activePneumococcal polypeptide or SEQ ID NO: 4 or an active variant orfragment thereof active variant or fragment thereof variant or fragmentthereof SEQ ID NO: 3 or an active Pneumolysoid or active variant or SEQID NO: 4 or an active variant or fragment thereof fragment thereofvariant or fragment thereof SEQ ID NO: 3 or an active L460D (SEQ ID NO:7 or 39 or SEQ ID NO: 4 or an active variant or fragment thereof activevariant or fragment thereof) variant or fragment thereof SEQ ID NO: 3 oran active Δ6N385 (SEQ ID NO: 8 or active SEQ ID NO: 4 or an activevariant or fragment thereof variant or fragment thereof) variant orfragment thereof SEQ ID NO: 3 or an active PdB (SEQ ID NO: 17 or activeSEQ ID NO: 4 or an active variant or fragment thereof variant orfragment thereof) variant or fragment thereof SEQ ID NO: 4 or an activePneumococcal polypeptide or SEQ ID NO: 1 or an active variant orfragment thereof active variant or fragment thereof variant or fragmentthereof SEQ ID NO: 4 or an active Pneumolysoid or active variant or SEQID NO: 1 or an active variant or fragment thereof fragment thereofvariant or fragment thereof SEQ ID NO: 4 or an active L460D (SEQ ID NO:7 or 39 or SEQ ID NO: 1 or an active variant or fragment thereof activevariant or fragment thereof) variant or fragment thereof SEQ ID NO: 4 oran active Δ6N385 (SEQ ID NO: 8 or active SEQ ID NO: 1 or an activevariant or fragment thereof variant or fragment thereof) variant orfragment thereof SEQ ID NO: 4 or an active PdB (SEQ ID NO: 17 or activeSEQ ID NO: 1 or an active variant or fragment thereof variant orfragment thereof) variant or fragment thereof SEQ ID NO: 4 or an activePneumococcal polypeptide or SEQ ID NO: 3 or an active variant orfragment thereof active variant or fragment thereof variant or fragmentthereof SEQ ID NO: 4 or an active Pneumolysoid or active variant or SEQID NO: 3 or an active variant or fragment thereof fragment thereofvariant or fragment thereof SEQ ID NO: 4 or an active L460D (SEQ ID NO:7 or 39 or SEQ ID NO: 3 or an active variant or fragment thereof activevariant or fragment thereof) variant or fragment thereof SEQ ID NO: 4 oran active Δ6N385 (SEQ ID NO: 8 or active SEQ ID NO: 3 or an activevariant or fragment thereof variant or fragment thereof) variant orfragment thereof SEQ ID NO: 4 or an active PdB (SEQ ID NO: 17 or activeSEQ ID NO: 3 or an active variant or fragment thereof variant orfragment thereof) variant or fragment thereof SEQ ID NO: 4 or an activePneumococcal polypeptide or SEQ ID NO: 4 or an active variant orfragment thereof active variant or fragment thereof variant or fragmentthereof SEQ ID NO: 4 or an active Pneumolysoid or active variant or SEQID NO: 4 or an active variant or fragment thereof fragment thereofvariant or fragment thereof SEQ ID NO: 4 or an active L460D (SEQ ID NO:7 or 39 or SEQ ID NO: 4 or an active variant or fragment thereof activevariant or fragment thereof) variant or fragment thereof SEQ ID NO: 4 oran active Δ6N385 (SEQ ID NO: 8 or active SEQ ID NO: 4 or an activevariant or fragment thereof variant or fragment thereof) variant orfragment thereof SEQ ID NO: 4 or an active PdB (SEQ ID NO: 17 or activeSEQ ID NO: 4 or an active variant or fragment thereof variant orfragment thereof) variant or fragment thereof *Table 1 denotes a fusionprotein with the first polypeptide fused in frame to the secondpolypeptide optionally fused in frame to the third polypeptide.Reference to active variants and fragments of SEQ ID NOS: 1, 2, 3 or 4in Table 1 further includes the polypeptide having a loop conformation.

B. Fusion Proteins Comprising Cytolysoids

As discussed above, the various CbpA fusion proteins provided herein caninclude a pneumolysoid polypeptide or active variant or fragment thereofto increase immunogenicity against pneumococcal infections. While CbpAis from pneumococcus it is recognized polypeptides from other type ofbacteria could be used to generate an immunogenic fusion protein whichcan produce protective antibodies against other forms of bacteria, forexample, bacteria from the genera Clostridium, Streptococcus, Listeria,Bacillus, and Arcanobacterium.

In one embodiment, the immunogenic fusion protein can comprise acytolysoid polypeptide or active variant or fragment thereof. As usedherein, a “cytolysoid fusion protein” can comprise a full lengthcytolysoid polypeptide or active variants or fragments thereof or anyimmunogenic fragment of cytolysoid as discussed in further detailelsewhere herein. Cytolysins are a family of pore-forming toxins thatare produced by more than 20 species from the genera Clostridium,Streptococcus, Listeria, Bacillus, and Arcanobacterium. Each cytolysinis produced as a monomer and upon encountering a eukaryotic cell themonomers convert into an oligomeric structure to form a pore complex.Cytolysins are well known as hemolytic proteins. As used herein,“cytolysoid” refers to a modified cytolysin, wherein the modification ofthe protein inactivates or reduces the oligomerization and/or hemolyticproperties of the cytolysoid protein while still retaining immunogenicactivity. A reduction in the toxicity of the cytolysin protein (i.e. areduction in oligomerization, and/or hemolysis) comprises at least a 1%,5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater statisticallysignificant decrease relative to an appropriate control. Various methodsto assay for cytolysin activity are known in the art and are the same asdescribed elsewhere herein for pneumolysin.

The art provides substantial guidance regarding the modificationsrequired to inactivate or reduce the toxic activity (i.e.oligomerization and/or hemolysis) of cytolysins. These modifications maybe amino acid substitutions, deletions, and/or additions. Suchmodifications are well known in the art. Some examples include, but arenot limited to, WO2005/108419 and WO2005/108580 which disclosecytolysoids having a mutation (e.g. a substitution or deletion) withinthe region corresponding to amino acids 144 to 161 of the wild-typepneumolysin protein. This region of pneumolysin has a consensus sequencethat is shared among the cytolysins. These mutant cytolysins havereduced oligomerization and/or hemolytic activity as compared to thewild-type cytolysin, and are therefore less toxic. The mutant may have asubstitution or deletion of one or more amino acids within the regionscorresponding to amino acids 144 to 161 of the wild-type pneumolysinsequence. Thus, the cytolysoid may have a mutation at one or more of theamino acids residues corresponding to amino acids 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161of wild-type pneumolysin. Additional, non-limiting, examples ofcytolysoids in the art are disclosed in U.S. Patent Application No.2009/0285846A1 and U.S. Patent Application No. 2010/0166795, which areherein incorporated by reference.

Any cytolysin can be modified to a cytolysoid and employed in the fusionproteins presented herein. Examples include, but are not limited to,pneumolysin from Streptococcus pneumoniae, perfringolysin O fromClostridium perfringens, intermedilysin from Streptococcus intermedius,alveolysin from Bacillus alvei, anthrolysin from Bacillus anthracis,putative cereolysin from Bacillus cereus, ivanolysin O from Listeriaivanovii, pyolysin from Arcanobacterium pyogenes, seeligeriolysin O fromListeria seeligeri, streptolysin O from S. pyogenes, suilysin fromStreptococcus suis, tetanolysin from Clostridium tetani, listeriolysin Ofrom Listeria monocytogenes, streptolysin O from Streptococcusequisimilis, streptolysin O from S. canis, thuringiolysin O fromBacillus thuringiensis, latersporolysin O from B. laterosporus,botulinolysin from Clostridium botulinum, chauveolysin from C. chauvoei,bifermentolysin from C. bifermentans, sordellilysin from C. sordellii,histolyticolysin from Clostridium histiolyticum, novylysin fromClostridium novyi, and septicolysin O from Clostridium septicum. Otherexamples of cytolysins and cytolysoids can be found, for example in S.E. Gelber et al. (2008) J. Bacteriology 190:3896-3903; and B. H. Jost etal. (2003) Infection and Immunity 71:2966-2969, herein incorporated byreference in their entirety.

The immunogenic cytolysoid fusion proteins provided herein can compriseat least 1, 2, 3, 4, 5 or more immunogenic bacterial polypeptides. Thebacterial polypeptide source can include, but is not limited to, theabove listed examples of cytolysin comprising bacteria. The immunogenicpolypeptides of the cytolysoid fusion proteins disclosed herein can beassembled in various combinations. The cytolysoid can be at either atthe N-terminal or C-terminal end of the fusion protein, or it can beflanked by immunogenic bacterial polypeptides. The immunogenic bacterialpolypeptides can be from the same bacteria as the cytolysoid or they canbe from different bacteria.

In a specific embodiment, the cytolysoid fusion protein comprises apneumolysoid (i.e. SEQ ID NOS: 7, 8, 17 or 39 or active variants orfragments thereof) and the immunogenic bacterial polypeptides cancomprise any immunogenic protein from pneumococcal bacteria.

Active variants or fragments of the various immunogenic cytolysoids areprovided herein. Such active variants can comprise at least 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to a cytolysoid polypeptide provided herein in thatthey maintain immunogenic activity, as described elsewhere herein.Active variants of immunogenic cytolysoids are known in the art. See,for example, U.S. Patent Application No. 2009/0285846A1 and U.S. PatentApplication No. 2010/0166795, herein incorporated by reference in theirentirety.

C. Polynucleotides Encoding the Immunogenic Fusion Proteins and Methodsof Making the Immunogenic Fusion Proteins

Compositions further include isolated polynucleotides that encode thevarious immunogenic fusion proteins described herein above, and variantsand fragments thereof. Exemplary polynucleotides comprising nucleotidesequences that encode the various polypeptides and the various fusionproteins are summarized in Table 4. Variants and fragments of theisolated polynucleotides disclosed herein are also encompassed.

Vectors and expression cassettes comprising the polynucleotidesdescribed herein are further disclosed. Expression cassettes willgenerally include a promoter operably linked to a polynucleotide and atranscriptional and translational termination region.

The use of the term “polynucleotide” is not intended to limit thepresent invention to polynucleotides comprising DNA. Those of ordinaryskill in the art will recognize that polynucleotides, can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues.

An “isolated” polynucleotide is substantially or essentially free fromcomponents that normally accompany or interact with the polynucleotideas found in its naturally occurring environment. Thus, an isolatedpolynucleotide is substantially free of other cellular material orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

Conventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art may be employed herein. Suchtechniques are explained fully in the literature. See, e.g., Sambrook etal., “Molecular Cloning: A Laboratory Manual” (1989); “Current Protocolsin Molecular Biology” Volumes 1-111 [Ausubel, R. M., ed. (1994)]; “CellBiology: A Laboratory Handbook” Volumes I-III [J. E. Celis, ed.(1994))]; “Current Protocols in Immunology” Volumes I-III [Coligan, J.E., ed. (1994)]; “Oligonucleotide Synthesis” (M. J. Gait ed. 1984);“Nucleic Acid Hybridization” [B. D. Hames & S. J. Higgins eds. (1985)];“Transcription And Translation” [B. D. Hames & S. J. Higgins, eds.(1984)]; “Animal Cell Culture” [R. I. Freshney, ed. (1986)];“Immobilized Cells And Enzymes” [IRL Press, (1986)]; B. Perbal, “APractical Guide To Molecular Cloning” (1984).

The polypeptides and fusion proteins disclosed herein may be altered invarious ways including amino acid substitutions, deletions, truncations,and insertions. Methods for such manipulations are generally known inthe art. For example, amino acid sequence variants and fragments of theCbpA or cytolysoid proteins can be prepared by mutations in the DNA.Methods for mutagenesis and polynucleotide alterations are well known inthe art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S.Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques inMolecular Biology (MacMillan Publishing Company, New York) and thereferences cited therein. In specific embodiments employing the loopedconformation of the R2₁ and R2₂ polypeptides, the mutation comprises atleast an insertion or a substitution of a cysteine residue in a CbpApolypeptide disclosed herein. In other embodiments, the mutations inCbpA (the R2 domain, the R2₁ or the R2₂ region) pneumolysin orcytolysins comprise at least a deletion, insertion, and/or amino acidsubstitution.

A vector which comprises the above-described polynucleotides operablylinked to a promoter is also provided herein. A nucleotide sequence is“operably linked” to an expression control sequence (e.g., a promoter)when the expression control sequence controls and regulates thetranscription and translation of that sequence. The term “operablylinked” when referring to a nucleotide sequence includes having anappropriate start signal (e.g., ATG) in front of the nucleotide sequenceto be expressed and maintaining the correct reading frame to permitexpression of the sequence under the control of the expression controlsequence and production of the desired product encoded by the sequence.If a gene that one desires to insert into a recombinant nucleic acidmolecule does not contain an appropriate start signal, such a startsignal can be inserted in front of the gene. A “vector” is a replicon,such as plasmid, phage or cosmid, to which another nucleic acid segmentmay be attached so as to bring about the replication of the attachedsegment. The promoter may be, or is identical to, a bacterial, yeast,insect or mammalian promoter. Further, the vector may be a plasmid,cosmid, yeast artificial chromosome (YAC), bacteriophage or eukaryoticviral DNA.

Other numerous vector backbones known in the art as useful forexpressing protein may be employed. Such vectors include, but are notlimited to: adenovirus, simian virus 40 (SV40), cytomegalovirus (CMV),mouse mammary tumor virus (MMTV), Moloney murine leukemia virus, DNAdelivery systems, i.e. liposomes, and expression plasmid deliverysystems. Further, one class of vectors comprises DNA elements derivedfrom viruses such as bovine papilloma virus, polyoma virus, baculovirus,retroviruses or Semliki Forest virus. Such vectors may be obtainedcommercially or assembled from the sequences described by methodswell-known in the art.

A host vector system for the production of a polypeptide which comprisesthe vector of a suitable host cell is provided herein. Suitable hostcells include, but are not limited to, prokaryotic or eukaryotic cells,e.g. bacterial cells (including gram positive cells), yeast cells,fungal cells, insect cells, and animal cells. Numerous mammalian cellsmay be used as hosts, including, but not limited to, the mousefibroblast cell NIH 3T3, CHO cells, HeLa cells, Ltk⁻ cells, etc.Additional animal cells, such as R1.1, B-W and L-M cells, African GreenMonkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insectcells (e.g., Sf9), and human cells and plant cells in tissue culture canalso be used.

A wide variety of host/expression vector combinations may be employed inexpressing the polynucleotide sequences presented herein. Usefulexpression vectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol El, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4;phage DNAS, e.g., the numerous derivatives of phage λ, e.g., NM989, andother phage DNA, e.g., M13 and filamentous single stranded phage DNA;yeast plasmids such as the 2μ plasmid or derivatives thereof; vectorsuseful in eukaryotic cells, such as vectors useful in insect ormammalian cells; vectors derived from combinations of plasmids and phageDNAs, such as plasmids that have been modified to employ phage DNA orother expression control sequences; and the like.

Any of a wide variety of expression control sequences (sequences thatcontrol the expression of a nucleotide sequence operably linked to it)may be used in these vectors to express the polynucleotide sequencesprovided herein. Such useful expression control sequences include, forexample, the early or late promoters of SV40, CMV, vaccinia, polyoma oradenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the LTR system, the major operator and promoter regions of phageλ, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase (e.g., Pho5), the promoters of the yeast α-matingfactors, and other sequences known to control the expression of genes ofprokaryotic or eukaryotic cells or their viruses, and variouscombinations thereof.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the polynucleotidesequences provided herein. Neither will all hosts function equally wellwith the same expression system. However, one skilled in the art will beable to select the proper vectors, expression control sequences, andhosts without undue experimentation to accomplish the desired expressionwithout departing from the scope of this invention. For example, inselecting a vector, the host must be considered because the vector mustfunction in it. The vector's copy number, the ability to control thatcopy number, and the expression of any other proteins encoded by thevector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular nucleotide sequence or gene to be expressed, particularlyas regards potential secondary structures. Suitable unicellular hostswill be selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the nucleotide sequencesto be expressed, and the ease of purification of the expressionproducts.

In preparing the expression cassette, the various polynucleotides may bemanipulated, so as to provide for the polynucleotide sequences in theproper orientation and, as appropriate, in the proper reading frame.Toward this end, adapters or linkers may be employed to join thepolynucleotides or other manipulations may be involved to provide forconvenient restriction sites, removal of superfluous DNA, removal ofrestriction sites, or the like. For example, linkers such as twoglycines may be added between polypeptides. Methionine residues encodedby atg nucleotide sequences may be added to allow initiation of genetranscription. For this purpose, in vitro mutagenesis, primer repair,restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

Further provided is a method of producing a polypeptide which comprisesexpressing a polynucleotide encoding a fusion protein disclosed hereinin a host cell under suitable conditions permitting the production ofthe polypeptide and recovering the polypeptide so produced.

D. Methods of Use

These fusion proteins disclosed herein comprising two or more distinctimmunogenic polypeptides represent a novel, cost effective, way toimprove vaccine efficacy. The CbpA, cytolysoid fusion proteins providedherein (such as those examples provided in Tables 1 and 2) areimmunogenic and depending on the design of the fusion protein and thechoice of the polypeptide components, they find use in the treatment andprevention of a variety of bacterial infections.

The compositions provided herein find use in methods for preventing andtreating bacterial infections. As used herein, “preventing a bacterialinfection” is intended administration of a therapeutically effectiveamount of an immunogenic fusion protein, immunogenic composition, orvaccine provided herein to an animal in order to protect the animal fromthe development of a bacterial infection or the symptoms thereof. Insome embodiments, a composition presented herein is administered to asubject, such as a human, that is at risk for developing a bacterialinfection. By “treating a bacterial infection” is intendedadministration of a therapeutically effective amount of a fusionprotein, immunogenic composition, or vaccine provided herein to ananimal that has a bacterial infection or that has been exposed to abacterium, where the purpose is to cure, heal, alleviate, relieve,alter, remedy, ameliorate, improve, or affect the condition or thesymptoms of the bacterial infection.

A “therapeutically effective amount” as used herein refers to thatamount which provides a therapeutic effect for a given condition andadministration regimen. Thus, the phrase “therapeutically effectiveamount” is used herein to mean an amount sufficient to cause animprovement in a clinically significant condition in the host. Inparticular aspects, a “therapeutically effective amount” refers to anamount of an immunogenic fusion protein, immunogenic composition, orvaccine provided herein that when administered to an animal brings abouta positive therapeutic response with respect to the prevention ortreatment of a subject for a bacterial infection. A positive therapeuticresponse with respect to preventing a bacterial infection includes, forexample, the production of antibodies by the subject in a quantitysufficient to protect against development of the disease. Similarly, apositive therapeutic response in regard to treating a bacterialinfection includes curing or ameliorating the symptoms of the disease.In the present context, a deficit in the response of the host can beevidenced by continuing or spreading bacterial infection. An improvementin a clinically significant condition in the host includes a decrease inbacterial load, clearance of bacteria from colonized host cells,reduction in fever or inflammation associated with infection, or areduction in any symptom associated with the bacterial infection.

In particular aspects, methods for preventing a pneumococcal infectionin an animal comprise administering to the animal a therapeuticallyeffective amount of an immunogenic fusion protein disclosed herein, animmunogenic composition comprising an immunogenic fusion proteindisclosed herein in combination with a pharmaceutically acceptablecarrier, or a vaccine disclosed herein, thereby preventing apneumococcal infection. When treating or preventing pneumococcalinfections, at least one of the various immunogenic fusion proteinscomprising at least one polypeptide from pneumococcus will be used(e.g., a CbpA fusion protein, a fusion peptide from any otherimmunogenic pneumococcal protein or a pneumolysoid fusion protein, asdiscussed elsewhere herein). In other embodiments, methods for treatinga pneumococcal infection in an animal infected with or exposed to apneumococcal bacterium comprise administering to the animal atherapeutically effective amount of a fusion protein, an immunogeniccomposition comprising a fusion protein in combination with apharmaceutically acceptable carrier, or a vaccine disclosed herein,thereby treating the animal. For example, in an individual alreadyinfected with a pneumococcal bacterium, an immunogenic fusion proteinprovided herein could be used as protection against the spread of theinfection from the blood to the brain.

A method of inducing an immune response in a subject which has beenexposed to or infected with a pneumococcal bacterium is further providedcomprising administering to the subject a therapeutically effectiveamount of an immunogenic fusion protein provided herein (i.e., such asthe fusion proteins listed in Tables 1 or 2), or a biologically activevariant or fragment thereof, an immunogenic composition, or a vaccine asdisclosed herein, thereby inducing an immune response.

Pneumococcal infection involves bacterial colonization of nasopharyngealepithelial cells and subsequent bacterial entry into the bloodstreamand, possibly, the brain. While not being bound by any theory, CbpAbinds to pTgR during colonization of the nasopharynx by pneumococcalbacteria and to the laminin receptor during the invasive phase of thedisease when the bacteria enter the bloodstream and the brain. The twobinding activities have been localized to specific regions of the R2domain of CbpA. In particular, the R2₁ region is responsible for bindingto pIgR and bacterial colonization in the nasopharynx, whereas the R2₂region is involved in binding to the laminin receptor and subsequentbacterial entry into the bloodstream and brain. This information can beutilized to develop immunogenic compositions and vaccines that areprotective against both steps of pneumococcal infection, namelycolonization of the nasopharynx and bacterial entry into thebloodstream.

In some embodiments, a fusion protein comprising, but not limited to, aCbpA polypeptide, or a biologically active variant or fragment thereof,can be employed in various methods to decrease pneumococcal colonizationof the nasopharynx (i.e. a fusion protein comprising the R2₁ region ofSEQ ID NOS: 1 or 3 or an active variant or fragment thereof, wherein theR2₁ region is in the loop conformation) or to decrease bacterial entryinto the bloodstream and brain (i.e. a fusion protein comprising the R2₂region of SEQ ID NOS: 2 or 4 or an active variant or fragment thereof,wherein said R2₂ region is in the loop conformation), or in otherembodiments, can be used to decrease bacterial entry into the lung, intothe bloodstream or across the blood brain barrier (i.e. a fusion proteincomprising both an R2₁ and R2₂ sequence such as those sequences of (SEQID NOS: 1, 2, 3 or 4, or active variants or fragments thereof, whereinthe R2₁ and/or the R2₂ are in the loop conformation). As used herein a“decrease” is meant at least a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% decrease relative to an appropriate control, oralternatively, decreased to a sufficient level to produce a desiredtherapeutic effect in the animal. Various methods to measure bacterialcolonization are known in the art. For example, bacteria in the bloodcan be measured by taking a blood sample and spreading the blood out onan agar plate which contains the appropriate medium for bacterialgrowth. Bacteria in the nasopharynx can be measured by culturingbacteria from a swab or lavage of the nasopharynx or lungs of an animal.Bacteria that have crossed the blood brain barrier can be measured in asample of cerebrospinal fluid or by detecting the physical attributes ofmeningitis in an animal, such as spinning of the head.

In further embodiments, a fusion protein comprising, but not limited to,any pneumococcal immunogenic polypeptide can be employed in variousmethods to treat and prevent pneumococcal infections.

In yet another embodiment, a fusion protein comprising a CbpApolypeptide provided herein can be employed in various methods to treatand prevent Neisseria meningitidis infection. Neisseria meningitidis isanother bacterium that crosses the blood brain barrier and causesmeningitis. As disclosed in U.S. Patent Publication No. 2010-0143394-A1,herein incorporated by reference, Neisseria meningitidis binds to thelaminin receptor to cross the blood brain barrier. This is the samemechanism used by Streptococcus pneumoniae. Herein, in Example 4, isdisclosed that fusion proteins comprising, but not limited to, the R2₁or R2₂ regions, R2₁ or R2₂ regions having loop conformations or activevariants or fragments thereof, of CbpA can cross-protect againstNeisseria meningitidis. Therefore, the fusion proteins provided hereinhave use as a vaccine for the treatment and prevention of infections ofother bacteria that utilize similar infectious mechanisms.

A fusion protein comprising a cytolysoid can be employed in variousmethods to treat and prevent bacterial infections. As discussed above,the cytolysoid polypeptides (or active variant or fragment thereof) canbe modified from any bacterial cytolysin and be employed to create afusion protein with one or more immunogenic polypeptides from the samebacterial source or a different bacterial source as the cytolysoid. Inthis way, methods to treat and prevent various bacterial infections areencompassed herein. Some examples of bacteria that may cause bacterialinfections are disclosed elsewhere herein.

The immunogenic fusion proteins provided herein could also be used invarious methods to treat or prevent multiple bacterial infections in ananimal. The immunogenic fusion proteins could comprise a combination ofimmunogenic polypeptides from two or more bacteria. In a particularaspect, the immunogenic polypeptides of the fusion protein wouldoriginate from bacterial sources that are frequently foundsimultaneously in a given animal. For example, infections caused byStreptococcus pneumoniae and Haemophilus influenzae, which cansimultaneously infect the nasopharynx, could be treated or prevented bya fusion protein comprising immunogenic polypeptides from both bacteria.

II. Pharmaceutical Compositions

Compositions further include immunogenic compositions and vaccinescomprising an immunogenic fusion protein disclosed herein. Immunogeniccompositions provided herein comprise at least one immunogenic fusionprotein as described herein in combination with a pharmaceuticallyacceptable carrier. In some embodiments, the fusion protein is presentin an amount effective to elicit antibody production when administeredto an animal. Methods for detecting antibody production in an animal arewell known in the art.

Vaccines for treating or preventing bacterial infection are provided andcomprise at least one fusion protein provided herein in combination witha pharmaceutically acceptable carrier, wherein the fusion protein ispresent in an amount effective for treating or preventing a bacterialinfection. In particular embodiments, the vaccine elicits production ofprotective antibodies against the bacteria when administered to ananimal. In specific embodiments, the vaccine comprises an immunogenicfusion protein comprising a cytolysoid. In other embodiments, thevaccine comprises an immunogenic fusion protein comprising a cytolysoidand one or more immunogenic polypeptides from the same bacterial sourceor a different bacterial source as the cytolysoid.

Vaccines for treating or preventing pneumococcal infection are alsoprovided and comprise at least one fusion protein provided herein incombination with a pharmaceutically acceptable carrier, wherein thefusion protein is present in an amount effective for treating orpreventing a pneumococcal infection. In particular embodiments, thevaccine elicits production of protective antibodies againstStreptococcus pneumoniae when administered to an animal. In specificembodiments, the vaccine comprises an immunogenic fusion proteincomprising a CbpA polypeptide(s) (i.e. such as those fusion proteinspresented in Table 1).

In addition, compositions comprising an immunogenic fusion protein orbiologically active variant or fragment thereof and an adjuvant incombination with a pharmaceutically acceptable carrier are provided. Theimmunogenic fusion proteins presented herein can be prepared in anadmixture with an adjuvant to prepare a vaccine. Pharmaceuticallyacceptable carriers and adjuvants are well known in the art. Methods forformulating pharmaceutical compositions and vaccines are generally knownin the art. A thorough discussion of formulation and selection ofpharmaceutical acceptable carriers, stabilizers, and isomolytes can befound in Remington's Pharmaceutical Sciences (18^(th) ed.; MackPublishing Company, Eaton, Pa., 1990), herein incorporated by reference.As provided herein, a vaccine may comprise, for example, at least one ofthe fusion proteins disclosed in Table 1 or a biologically activevariant or fragment thereof.

As described elsewhere herein, the R2₁ region of CbpA is believed to beinvolved in bacterial colonization of the nasopharynx and the R2₂ regionof CbpA mediates bacterial entry into the bloodstream. Thus a vaccinethat comprises a fusion protein comprising both an R2₁ and an R2₂polypeptide can provide protection against both steps involved inpneumococcal infection. In specific embodiments, a vaccine comprising afusion protein comprising both an R2₁ and an R2₂ polypeptide, forexample, the fusion protein of SEQ ID NO: 9 or active variants orfragments thereof, may provide protection against both steps involved inpneumococcal infection.

The immunogenic compositions and vaccines disclosed herein may furthercomprise a mixture of 1 or more fusion proteins with 1 or morepolypeptides provided herein. A vaccine may comprise, for example, anyone of the immunogenic fusion proteins described in Table 1 or activevariants or fragments thereof combined as a mixture with one or more ofthe polypeptides set forth in SEQ ID NOS: 1, 2, 3, 4, 5, 7, 8, 12, 13,14, 17 or 39 or active variants or fragments thereof.

Further provided is a vaccine for treating or preventing a Neisseriameningitidis infection comprising an immunogenic fusion proteindisclosed herein (i.e. such as the fusion proteins presented in Table 1)and a pharmaceutically acceptable carrier. As disclosed elsewhereherein, fusion proteins comprising, but not limited to, the R2₁ or R2₂regions, R2₁ or R2₂ regions having loop conformations or active variantsor fragments thereof, of CbpA can cross-protect against Neisseriameningitidis.

III. Methods of Administration

The vaccines provided herein can be administered via any parenteralroute, including, but not limited, to intramuscular, intraperitoneal,intravenous, and the like. Preferably, since the desired result ofvaccination is to elucidate an immune response to the antigen, andthereby to the pathogenic organism, administration directly, or bytargeting or choice of a viral vector, indirectly, to lymphoid tissues,e.g., lymph nodes or spleen, is desirable. Since immune cells arecontinually replicating, they are ideal targets for retroviralvector-based nucleic acid vaccines, since retroviruses requirereplicating cells.

Further, as used herein “pharmaceutically acceptable carrier” are wellknown to those skilled in the art and include, but are not limited to,0.01-0.1 M and preferably 0.05M phosphate buffer or 0.8% saline.Additionally, such pharmaceutically acceptable carriers may be aqueousor non-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, antimicrobials, antioxidants, collating agents, inertgases and the like.

The term “adjuvant” refers to a compound or mixture that enhances theimmune response to an antigen. An adjuvant can serve as a tissue depotthat slowly releases the antigen and also as a lymphoid system activatorthat non-specifically enhances the immune response (Hood et al.,Immunology, Second Ed., 1984, Benjamin/Cummings: Menlo Park, Calif., p.384). Often, a primary challenge with an antigen alone, in the absenceof an adjuvant, will fail to elicit a humoral or cellular immuneresponse. Adjuvant include, but are not limited to, complete Freund'sadjuvant, incomplete Freund's adjuvant, saponin, mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvant such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum.

Controlled or sustained release compositions include formulation inlipophilic depots (e.g. fatty acids, waxes, oils). Also comprehendedherein are particulate compositions coated with polymers (e.g.poloxamers or poloxamines) and the compound coupled to antibodiesdirected against tissue-specific receptors, ligands or antigens orcoupled to ligands of tissue-specific receptors. Other embodiments ofthe compositions presented herein incorporate particulate formsprotective coatings, protease inhibitors or permeation enhancers forvarious routes of administration, including parenteral, pulmonary, nasaland oral.

When administered, compounds are often cleared rapidly from mucosalsurfaces or the circulation and may therefore elicit relativelyshort-lived pharmacological activity. Consequently, frequentadministrations of relatively large doses of bioactive compounds may byrequired to sustain therapeutic efficacy. Compounds modified by thecovalent attachment of water-soluble polymers such as polyethyleneglycol, copolymers of polyethylene glycol and polypropylene glycol,carboxymethyl cellulose, dextran, polyvinyl alcohol,polyvinylpyrrolidone or polyproline are known to exhibit substantiallylonger half-lives in blood following intravenous injection than do thecorresponding unmodified compounds (Abuchowski et al., 1981; Newmark etal., 1982; and Katre et al., 1987). Such modifications may also increasethe compound's solubility in aqueous solution, eliminate aggregation,enhance the physical and chemical stability of the compound, and greatlyreduce the immunogenicity and reactivity of the compound. As a result,the desired in vivo biological activity may be achieved by theadministration of such polymer-compound abducts less frequently or inlower doses than with the unmodified compound.

Dosages.

The sufficient amount may include but is not limited to from about 1μg/kg to about 1000 mg/kg. The amount may be 10 mg/kg. Thepharmaceutically acceptable form of the composition includes apharmaceutically acceptable carrier.

The preparation of therapeutic compositions which contain an activecomponent is well understood in the art. Typically, such compositionsare prepared as an aerosol of the polypeptide delivered to thenasopharynx or as injectables, either as liquid solutions orsuspensions, however, solid forms suitable for solution in, orsuspension in, liquid prior to injection can also be prepared. Thepreparation can also be emulsified. The active therapeutic ingredient isoften mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents which enhance the effectivenessof the active ingredient.

An active component can be formulated into the therapeutic compositionas neutralized pharmaceutically acceptable salt forms. Pharmaceuticallyacceptable salts include the acid addition salts (formed with the freeamino groups of the polypeptide) and which are formed with inorganicacids such as, for example, hydrochloric or phosphoric acids, or suchorganic acids as acetic, oxalic, tartaric, mandelic, and the like. Saltsformed from the free carboxyl groups can also be derived from inorganicbases such as, for example, sodium, potassium, ammonium, calcium, orferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The component or components of a therapeutic composition provided hereinmay be introduced parenterally, transmucosally, e.g., orally, nasally,pulmonarily, or rectally, or transdermally. Preferably, administrationis parenteral, e.g., via intravenous injection, and also including, butis not limited to, intra-arteriole, intramuscular, intradermal,subcutaneous, intraperitoneal, intraventricular, and intracranialadministration. Oral or pulmonary delivery may be preferred to activatemucosal immunity; since pneumococci generally colonize thenasopharyngeal and pulmonary mucosa, mucosal immunity may be aparticularly effective preventive treatment. The term “unit dose” whenused in reference to a therapeutic composition provided herein refers tophysically discrete units suitable as unitary dosage for humans, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired diluent; i.e., carrier, or vehicle.

In another embodiment, the active compound can be delivered in avesicle, in particular a liposome (see Langer (1990) Science249:1527-1533; Treat et al., in Liposomes in the Therapy of InfectiousDisease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York,pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid).

In yet another embodiment, the therapeutic compound can be delivered ina controlled release system. For example, the fusion protein may beadministered using intravenous infusion, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. In oneembodiment, a pump may be used (see Langer, supra; Sefton (1987) CRCCrit. Ref Biomed. Eng. 14:201; Buchwald et al. (1980) Surgery 88:507;Saudek et al. (1989) N. Engl. J. Med. 321:574). In another embodiment,polymeric materials can be used (see Medical Applications of ControlledRelease, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974);Controlled Drug Bioavailability, Drug Product Design and Performance,Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas (1983)J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al.(1985) Science 228:190; During et al. (1989) Ann. Neurol. 25:351; Howardet al. (1989) J. Neurosurg. 71:105). In yet another embodiment, acontrolled release system can be placed in proximity of the therapeutictarget, i.e., the brain, thus requiring only a fraction of the systemicdose (see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems arediscussed in the review by Langer (1990) Science 249:1527-1533.

A subject in whom administration of an active component as set forthabove is an effective therapeutic regimen for a bacterial infection ispreferably a human, but can be any animal. Thus, as can be readilyappreciated by one of ordinary skill in the art, the methods andpharmaceutical compositions provided herein are particularly suited toadministration to any animal, particularly a mammal, and including, butby no means limited to, domestic animals, such as feline or caninesubjects, farm animals, such as but not limited to bovine, equine,caprine, ovine, and porcine subjects, wild animals (whether in the wildor in a zoological garden), research animals, such as mice, rats,rabbits, goats, sheep, pigs, dogs, cats, etc., i.e., for veterinarymedical use.

In the therapeutic methods and compositions provided herein, atherapeutically effective dosage of the active component is provided. Atherapeutically effective dosage can be determined by the ordinaryskilled medical worker based on patient characteristics (age, weight,sex, condition, complications, other diseases, etc.), as is well knownin the art. Furthermore, as further routine studies are conducted, morespecific information will emerge regarding appropriate dosage levels fortreatment of various conditions in various patients, and the ordinaryskilled worker, considering the therapeutic context, age and generalhealth of the recipient, is able to ascertain proper dosing. Generally,for intravenous injection or infusion, dosage may be lower than forintraperitoneal, intramuscular, or other route of administration. Thedosing schedule may vary, depending on the circulation half-life, andthe formulation used. The compositions are administered in a mannercompatible with the dosage formulation in the therapeutically effectiveamount. Precise amounts of active ingredient required to be administereddepend on the judgment of the practitioner and are peculiar to eachindividual. However, suitable dosages may range from about 0.1 to 20,preferably about 0.5 to about 10, and more preferably one to several,milligrams of active ingredient per kilogram body weight of individualper day and depend on the route of administration. Suitable regimes forinitial administration and booster shots are also variable, but aretypified by an initial administration followed by repeated doses at oneor more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations of ten nanomolar to ten micromolarin the blood are contemplated.

Administration with Other Compounds.

For treatment of a bacterial infection, one may administer the presentactive component in conjunction with one or more pharmaceuticalcompositions used for treating bacterial infection, including but notlimited to (1) antibiotics; (2) soluble carbohydrate inhibitors ofbacterial adhesin; (3) other small molecule inhibitors of bacterialadhesin; (4) inhibitors of bacterial metabolism, transport, ortransformation; (5) stimulators of bacterial lysis, or (6)anti-bacterial antibodies or vaccines directed at other bacterialantigens. Other potential active components include anti-inflammatoryagents, such as steroids and non-steroidal anti-inflammatory drugs.Administration may be simultaneous (for example, administration of amixture of the present active component and an antibiotic), or may be inseriatim.

Also contemplated herein is pulmonary or intranasal delivery of thepresent fusion protein (or derivatives thereof). The fusion protein (orderivative) is delivered to the lungs of a mammal, where it caninterfere with bacterial, i.e., streptococcal, and preferablypneumococcal binding to host cells. Other reports of preparation ofproteins for pulmonary delivery are found in the art [Adjei et al.(1990) Pharmaceutical Research, 7:565-569; Adjei et al. (1990)International Journal of Pharmaceutics, 63:135-144 (leuprolide acetate);Braquet et al. (1989) Journal of Cardiovascular Pharmacology, 13 (suppl.5): 143-146 (endothelin-1); Hubbard et al. (1989) Annals of InternalMedicine, Vol. III, pp. 206-212 (α1-antitrypsin); Smith et al. (1989) J.Clin. Invest. 84:1145-1146 (α-1-proteinase); Oswein et al.,“Aerosolization of Proteins”, Proceedings of Symposium on RespiratoryDrug Delivery II, Keystone, Colo., March, (1990) (recombinant humangrowth hormone); Debs et al. (1988) J. Immunol. 140:3482-3488(interferon-γ and tumor necrosis factor alpha); Platz et al., U.S. Pat.No. 5,284,656 (granulocyte colony stimulating factor)]. A method andcomposition for pulmonary delivery of drugs is described in U.S. Pat.No. 5,451,569, issued Sep. 19, 1995 to Wong et al.

All such devices require the use of formulations suitable for thedispensing of a fusion protein provided herein (or derivative).Typically, each formulation is specific to the type of device employedand may involve the use of an appropriate propellant material, inaddition to the usual diluents, adjuvant and/or carriers useful intherapy. Also, the use of liposomes, microcapsules or microspheres,inclusion complexes, or other types of carriers is contemplated.Chemically modified fusion proteins may also be prepared in differentformulations depending on the type of chemical modification or the typeof device employed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise at least one fusion protein (orderivative) dissolved in water at a concentration of about 0.1 to 25 mgof biologically active fusion protein per ml of solution. Theformulation may also include a buffer and a simple sugar (e.g., forstabilization and regulation of osmotic pressure). The nebulizerformulation may also contain a surfactant, to reduce or prevent surfaceinduced aggregation of the polypeptide caused by atomization of thesolution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the fusion protein (orderivative) suspended in a propellant with the aid of a surfactant. Thepropellant may be any conventional material employed for this purpose,such as a chlorofluorocarbon, a hydrochlorofluorocarbon, ahydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol, and1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactantsinclude sorbitan trioleate and soya lecithin. Oleic acid may also beuseful as a surfactant.

The liquid aerosol formulations contain a fusion protein provided hereinand a dispersing agent in a physiologically acceptable diluent. The drypowder aerosol formulations consist of a finely divided solid form of afusion protein provided herein and a dispersing agent. With either theliquid or dry powder aerosol formulation, the formulation must beaerosolized. That is, it must be broken down into liquid or solidparticles in order to ensure that the aerosolized dose actually reachesthe mucous membranes of the nasal passages or the lung. The term“aerosol particle” is used herein to describe the liquid or solidparticle suitable for nasal or pulmonary administration, i.e., that willreach the mucous membranes. Other considerations, such as constructionof the delivery device, additional components in the formulation, andparticle characteristics are important. These aspects of pulmonaryadministration of a drug are well known in the art, and manipulation offormulations, aerosolization means and construction of a delivery devicerequire at most routine experimentation by one of ordinary skill in theart. In a particular embodiment, the mass median dynamic diameter willbe 5 micrometers or less in order to ensure that the drug particlesreach the lung alveoli [Wearley, L. L. (1991) Crit. Rev. in Ther. DrugCarrier Systems 8:333].

Systems of aerosol delivery, such as the pressurized metered doseinhaler and the dry powder inhaler are disclosed in Newman, S. P.,Aerosols and the Lung, Clarke, S. W. and Davia, D. editors, pp. 197-22and can be used herein.

In a further embodiment, as discussed in detail infra, an aerosolformulation can include other therapeutically or pharmacologicallyactive ingredients in addition to a fusion protein, such as but notlimited to an antibiotic, a steroid, a non-steroidal anti-inflammatorydrug, etc.

Liquid Aerosol Formulations.

Also provided are aerosol formulations and dosage forms for use intreating subjects suffering from bacterial, e.g., streptococcal, inparticularly pneumococcal, infection. In general such dosage formscontain at least one fusion protein in a pharmaceutically acceptablediluent. Pharmaceutically acceptable diluents include but are notlimited to sterile water, saline, buffered saline, dextrose solution,and the like. In a specific embodiment, a diluent that may be used inthe pharmaceutical formulation provided herein is phosphate bufferedsaline or a buffered saline solution generally between the pH 7.0-8.0range, or water.

The liquid aerosol formulation provided herein may include, as optionalingredients, pharmaceutically acceptable carriers, diluents,solubilizing or emulsifying agents, surfactants and excipients. Theformulation may include a carrier. The carrier is a macromolecule whichis soluble in the circulatory system and which is physiologicallyacceptable where physiological acceptance means that those of skill inthe art would accept injection of said carrier into a patient as part ofa therapeutic regime. The carrier preferably is relatively stable in thecirculatory system with an acceptable plasma half life for clearance.Such macromolecules include but are not limited to Soya lecithin, oleicacid and sorbitan trioleate, with sorbitan trioleate preferred.

The formulations of the present embodiment may also include other agentsuseful for pH maintenance, solution stabilization, or for the regulationof osmotic pressure. Examples of the agents include but are not limitedto salts, such as sodium chloride, or potassium chloride, andcarbohydrates, such as glucose, galactose or mannose, and the like.

Further contemplated are liquid aerosol formulations comprising at leastone fusion protein and another therapeutically effective drug, such asan antibiotic, a steroid, a non-steroidal anti-inflammatory drug, etc.

Aerosol Dry Powder Formulations.

It is also contemplated that the present aerosol formulation can beprepared as a dry powder formulation comprising a finely divided powderform of fusion proteins and a dispersant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing at least one fusion protein (orderivative) and may also include a bulking agent, such as lactose,sorbitol, sucrose, or mannitol in amounts which facilitate dispersal ofthe powder from the device, e.g., 50 to 90% by weight of theformulation. The fusion protein (or derivative) should mostadvantageously be prepared in particulate form with an average particlesize of less than 10 mm (or microns), most preferably 0.5 to 5 mm, formost effective delivery to the distal lung. In another embodiment, thedry powder formulation can comprise a finely divided dry powdercontaining a fusion protein, a dispersing agent and also a bulkingagent. Bulking agents useful in conjunction with the present formulationinclude such agents as lactose, sorbitol, sucrose, or mannitol, inamounts that facilitate the dispersal of the powder from the device.

Also contemplated are dry powder formulations comprising at least onefusion protein and another therapeutically effective drug, such as anantibiotic, a steroid, a non-steroidal anti-inflammatory drug, etc.

Contemplated for use herein are oral solid dosage forms, which aredescribed generally in Remington's Pharmaceutical Sciences, 18th Ed.1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which isherein incorporated by reference. Solid dosage forms include tablets,capsules, pills, troches or lozenges, cachets or pellets. Also,liposomal or proteinoid encapsulation may be used to formulate thepresent compositions (as, for example, proteinoid microspheres reportedin U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and theliposomes may be derivatized with various polymers (e.g., U.S. Pat. No.5,013,556). A description of possible solid dosage forms for thetherapeutic is given by Marshall, K. In: Modern Pharmaceutics Edited byG. S. Banker and C. T. Rhodes Chapter 10, 1979, herein incorporated byreference. In general, the formulation will include the component orcomponents (or chemically modified forms thereof) and inert ingredientswhich allow for protection against the stomach environment, and releaseof the biologically active material in the intestine.

Also specifically contemplated are oral dosage forms of the abovederivatized component or components. The component or components may bechemically modified so that oral delivery of the derivative isefficacious. Generally, the chemical modification contemplated is theattachment of at least one moiety to the component molecule itself,where said moiety permits (a) inhibition of proteolysis; and (b) uptakeinto the blood stream from the stomach or intestine. Also desired is theincrease in overall stability of the component or components andincrease in circulation time in the body. Examples of such moietiesinclude: polyethylene glycol, copolymers of ethylene glycol andpropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol,polyvinyl pyrrolidone and polyproline. Abuchowski and Davis (1981)“Soluble Polymer-Enzyme Abducts” In: Enzymes as Drugs, Hocenberg andRoberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383; Newmark,et al. (1982) J. Appl. Biochem. 4:185-189. Other polymers that could beused are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred forpharmaceutical usage, as indicated above, are polyethylene glycolmoieties.

For the component (or derivative) the location of release may be thestomach, the small intestine (the duodenum, the jejunem, or the ileum),or the large intestine. One skilled in the art has availableformulations which will not dissolve in the stomach, yet will releasethe material in the duodenum or elsewhere in the intestine. Preferably,the release will avoid the deleterious effects of the stomachenvironment, either by protection of the protein (or derivative) or byrelease of the biologically active material beyond the stomachenvironment, such as in the intestine.

To ensure full gastric resistance a coating impermeable to at least pH5.0 is essential. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings which make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic i.e. powder; for liquid forms, a soft gelatin shell may beused. The shell material of cachets could be thick starch or otheredible paper. For pills, lozenges, molded tablets or tablet triturates,moist massing techniques can be used.

The peptide therapeutic can be included in the formulation as finemultiparticulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, theprotein (or derivative) may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

One may dilute or increase the volume of the therapeutic with an inertmaterial. These diluents could include carbohydrates, especiallymannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modifieddextran and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex,STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrates include but are notlimited to starch, including the commercial disintegrant based onstarch, Explotab. Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. Another form of the disintegrants are the insolublecationic exchange resins. Powdered gums may be used as disintegrants andas binders and these can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants. Binders may be used to hold the therapeutic agenttogether to form a hard tablet and include materials from naturalproducts such as acacia, tragacanth, starch and gelatin. Others includemethyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose(CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose(HPMC) could both be used in alcoholic solutions to granulate thetherapeutic.

An antifrictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants may include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment asurfactant might be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents mightbe used and could include benzalkonium chloride or benzethomiumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios.

Additives which potentially enhance uptake of the fusion protein (orderivative) are for instance the fatty acids oleic acid, linoleic acidand linolenic acid.

Pulmonary Delivery.

Also contemplated herein is pulmonary delivery of the present fusionprotein (or derivatives thereof). The fusion protein (or derivative) isdelivered to the lungs of a mammal while inhaling and coats the mucosalsurface of the alveoli. Other reports of this include Adjei et al.(1990) Pharmaceutical Research 7:565-569; Adjei et al. (1990)International Journal of Pharmaceutics 63:135-144 (leuprolide acetate);Braquet et al. (1989) Journal of Cardiovascular Pharmacology 13 (suppl.5): 143-146 (endothelin-1); Hubbard et al. (1989) Annals of InternalMedicine Vol. III, pp. 206-212 (a1-antitrypsin); Smith et al. (1989) J.Clin. Invest. 84:1145-1146 (a-1-proteinase); Oswein et al. (1990)“Aerosolization of Proteins”, Proceedings of Symposium on RespiratoryDrug Delivery II, Keystone, Colo., March, (recombinant human growthhormone); Debs et al. (1988) J. Immunol. 140:3482-3488 (interferon-g andtumor necrosis factor alpha) and Platz et al., U.S. Pat. No. 5,284,656(granulocyte colony stimulating factor). A method and composition forpulmonary delivery of drugs for systemic effect is described in U.S.Pat. No. 5,451,569, issued Sep. 19, 1995 to Wong et al.

Contemplated for use are a wide range of mechanical devices designed forpulmonary delivery of therapeutic products, including but not limited tonebulizers, metered dose inhalers, and powder inhalers, all of which arefamiliar to those skilled in the art.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise fusion protein (or derivative)dissolved in water at a concentration of about 0.1 to 25 mg ofbiologically active protein per ml of solution. The formulation may alsoinclude a buffer and a simple sugar (e.g., for protein stabilization andregulation of osmotic pressure). The nebulizer formulation may alsocontain a surfactant, to reduce or prevent surface induced aggregationof the protein caused by atomization of the solution in forming theaerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the fusion protein (orderivative) suspended in a propellant with the aid of a surfactant. Thepropellant may be any conventional material employed for this purpose,such as a chlorofluorocarbon, a hydrochlorofluorocarbon, ahydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol, and1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactantsinclude sorbitan trioleate and soya lecithin. Oleic acid may also beuseful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing fusion protein (or derivative) andmay also include a bulking agent, such as lactose, sorbitol, sucrose, ormannitol in amounts which facilitate dispersal of the powder from thedevice, e.g., 50 to 90% by weight of the formulation. The fusion protein(or derivative) should most advantageously be prepared in particulateform with an average particle size of less than 10 mm (or microns), mostpreferably 0.5 to 5 mm, for most effective delivery to the distal lung.

Nasal Delivery.

Nasal or nasopharyngeal delivery of the fusion protein (or derivative)is also contemplated. Nasal delivery allows the passage of the fusionprotein directly over the upper respiratory tract mucosal afteradministering the therapeutic product to the nose, without the necessityfor deposition of the product in the lung. Formulations for nasaldelivery include those with dextran or cyclodextran.

IV. Variants and Fragments of the Disclosed Polynucleotides andPolypeptides

Active variants and fragments of the disclosed polynucleotides andpolypeptides are also employed in the immunogenic fusion proteinsdescribed herein. “Variants” refer to substantially similar sequences.As used herein, a “variant polypeptide” is intended to mean apolypeptide derived from the native protein by deletion (so-calledtruncation) of one or more amino acids at the N-terminal and/orC-terminal end of the native protein; deletion and/or addition of one ormore amino acids at one or more internal sites in the native protein; orsubstitution of one or more amino acids at one or more sites in thenative protein. Variant polypeptides continue to possess the desiredbiological activity of the native polypeptide, that is, they areimmunogenic. A variant of an polypeptide or polynucleotide sequencedisclosed herein (i.e. SEQ ID NOS: 1-25 or 39) will typically have atleast about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity with the reference sequence.

The term “fragment” refers to a portion of an amino acid or nucleotidesequence comprising a specified number of contiguous amino acid ornucleotide residues. In particular embodiments, a fragment of apolypeptide disclosed herein may retain the biological activity of thefull-length polypeptide and hence be immunogenic. Fragments of apolynucleotide may encode protein fragments that retain the biologicalactivity of the protein and hence be immunogenic. Alternatively,fragments of a polynucleotide that are useful as PCR primers generallydo not retain biological activity. Thus, fragments of a nucleotidesequence disclosed herein (i.e. SEQ ID NOS: 6 or 10) may range from atleast about 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 175, 200, 225, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1100,1200, 1300, 1400, or 1500 contiguous nucleotides or up to thefull-length polynucleotide. Fragments of a polypeptide sequencedisclosed herein (i.e. SEQ ID NOS: 1-5, 7-9, 11, 12-14, 17-25 or 39) maycomprise at least 10, 15, 25, 30, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 400, 425,450, 475, or 500 contiguous amino acids, or up to the total number ofamino acids present in a full-length protein.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The BLAST programs of Altschul et al (1990) J. Mol. Biol. 215:403 arebased on the algorithm of Karlin and Altschul (1990) supra. BLASTnucleotide searches can be performed with the BLASTN program, score=100,wordlength=12, to obtain nucleotide sequences homologous to a nucleotidesequence provided herein. To obtain gapped alignments for comparisonpurposes, Gapped BLAST (in BLAST 2.0) can be utilized as described inAltschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively,PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search thatdetects distant relationships between molecules. See Altschul et al.(1997) supra. When utilizing BLAST, Gapped BLAST, PST-BLAST, the defaultparameters of the respective programs (e.g., BLASTN for nucleotidesequences, BLASTX for proteins) can be used. Sec www.ncbi.nlm.nih.gov.Alignment may also be performed manually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix. By “equivalent program” is intended anysequence comparison program that, for any two sequences in question,generates an alignment having identical nucleotide or amino acid residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

Units, prefixes, and symbols may be denoted in their ST accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges are inclusiveof the numbers defining the range. Amino acids may be referred to hereinby either their commonly known three letter symbols or by the one-lettersymbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes. The above-defined terms are more fullydefined by reference to the specification as a whole.

The following examples are provided by way of illustration, not by wayof limitation.

EXPERIMENTAL Background

Infection by S. pneumoniae remains a significant health threatworldwide. Shortcomings of the current vaccines include: restrictedprotective activity based on serotype, <50% protection againstpneumonia, and production costs that are too high for developing worlduse. A new vaccine must be protein-based to be immunogenic in high-riskchildren under the age of 2 yrs. Thus, candidate vaccines should becomposed of highly conserved proteins, preferably whose function isimportant in disease at multiple stages of infection. We have designed avaccine composed of pneumolysoid (toxoid of pneumolysin) and CbpA. Theseproteins elicit some of the highest antibody titers in sera from humansexposed to pneumococci (McCool, T. et al. Infect Immun 71, 5724-5732(2003)), their functions in disease pathogenesis are known, and involveprocesses in all body compartments (Rosenow, C. et al. Mol Microbiol 25,819-829 (1997); Watson, D. et al. Eur J Clin Microbiol Infect Dis 195,479-490 (1995)). There is strong preclinical data in animals suggestingprotective activity across serotypes, and they are even more efficaciouswhen used in combinations with each other (Palaniappan, R. et al. InfectImmun 73, 1006-1013 (2005); Briles, D. et al. J Infect Dis 188, 339-348(2003); Ogunniyi, A., Woodrow, M., Poolman, J. & Paton, J. Infect Immun69, 5997-6003 (2001)). To minimize cost but still provide efficacy to abreadth of serotypes, we have used genetic fusions of key components ofthese three proteins. Pneumolysoid has been used as a scaffold to whichcomponents of CbpA have been added.

Example 1: CbpA Peptide Fusions to PdB Pneumolysoid

Aim:

Construct and test a vaccine where genes encoding CbpA peptides arefused to the gene for pneumolysoid and the resulting single polypeptidehas CbpA and pneumolysoid domains.

Conclude:

Peptide fusion is immunogenic for both toxoid and CbpA domains; Linearpeptide fusion is not protective; looped peptide fusion is protective.

Significance:

CbpA peptide elicits antibody in the context of fusion to a toxoid andthe antibody is protective and the tertiary looped structure of thepeptide is preserved.

Background for Fusion of Proteins to CbpA Peptides

CbpA contains a region of important biological activity, termed R2 (SEQID NO: 14) which can be subdivided into two bioactive fragments YPT (R2₁region) and NEEK (R2₂ region). These regions are shown in FIGS. 1 and 2.US 2010-0143394-A1 shows how these two regions can be used as vaccinesand elicit the full protection that the entire CbpA protein confers. Asshown in US 2010-0143394-A1 (FIG. 1), small peptides such as from theR2₁ or R2₂ regions are not recognized by the immune system and thereforedo not generate a protective response when used alone as vaccines in amouse model of pneumococcal infection. This is true even if the peptideis modified to be held in the appropriate folded tertiary conformation.

Also as shown in US 2010-0143394-A1, attaching the peptide to a proteinmakes the peptides part of a bigger molecule and it becomes detectableby the immune system and generates antibodies. Such proteins are“carriers” for the peptides. The carrier protein call be a T cellepitope (TCE) of the sequence listed in SEQ ID NO: 15 or 16 or a largerprotein containing TCEs. Care must be taken to maintain the native threedimensional conformation of all the components in a fusion polypeptideso they do not interfere with each other and generate antibodies thatrecognize the conformation of the native protein from which the piececame.

Construction of Fusions Between CbpA Peptides and Pneumolysin Toxoid PdB

Since combinations of proteins can be more effective vaccines thansingle proteins alone but vaccines with more components are moreexpensive, we created genetic constructs where the gene sequences forthe bioactive fragments of CbpA were linked directly to the gene forpneumolysoid PdB (detoxified pneumolysin). This is termed a ‘fusion’.Upon transcription/translation, one protein is made from the fused geneparts. In this way, the important components of several proteins can beincorporated into one polypeptide.

The peptides of CbpA that are of particular interest are shown in FIG. 2in boxes labeled YPT and NEEK (SEQ ID NOS: 1-4). This set of experimentstests two properties of the fusion of YPT and/or NEEK to pneumolysoidPdB. First (part 1) it tests if the small peptide fused to the largerPdB can be detected by the immune system, i.e. make antibody. Second(part 2) tests if the conformation of the YPT and/or NEEK as eitherlinear or looped (native conformation) makes a difference in theprotective activity of the immune response.

Two constructs were made and tested in experiments part 1 and 2:

Construct 1: Linear NEEK fused to PdB: The carrier protein pneumolysoid,PdB, was compared to PdB-2-TCEs-linear-NEEK (SEQ ID NO: 18), a constructmodified to fuse a TCE and linear NEEK to the C terminus. The linearfragment is the native sequence but when not within the native proteinit unfolds and becomes linear rather than looped as shown in FIG. 2.This linear form does not generate antibody to the native CbpA andtherefore is not expected to be protective. Therefore, this constructtests if fusing any protein to pneumolysoid can generate specificantibodies to both the PdB and the peptide.Construct 2: Looped YPT added to construct 1: This construct adds thelooped YPT to construct 1 and tests if the looped YPT generates antibodyand if that antibody is protective in the context of PdB and linearNEEK. The carrier protein pneumolysoid, PdB, was compared tolooped-YPT-PdB-2TCEs-linear-NEEK (SEQ ID NO: 19), a construct modifiedto fuse a looped YPT domain on the N-terminus and a TCE with linear NEEKto the C-terminus.

Construct 1:

Primers were designed to add 2 tandem T-cell epitopes (TCE's) and linearNEEK sequence to PdB Pneumolysin toxoid. (This NEEK was linear andlacked cysteine linkers which allow loop formation.) All fusion peptidesare linked together with two glycine residues denoted by the lowercaseletters. Restriction sites are underlined.

Primers:

FORWARD JAT 201 (SEQ ID NO: 26) 5′-CgCgGGATCCAGAAGATGGCAAATAAAGCAG-3′REVERSE C-Term Fusion (SEQ ID NO: 27)5′-CgCgGAGCTCAGAGCTATTTAAGTTGCTTAACTTTTTCCTCGTTTCGAGGTTCCTTAGCAAGTTTaccaccAGTAATACCAATAAATTTAGAATTAGCTTTAATATATTGAGTAATACCAATAAATTTAGAATTAGCTTTAATATATTGaccaccGTCATTTTCTACCTTATCTTCTACC-3′Recloned into pET15b with primers:

JAT209 (SEQ ID NO: 28) 5′-CGCGCATATGAAGATGGCAAATAAAGCAG-3′ JAT210(SEQ ID NO: 29) 5′-CGCGGGATCCAGAGCTATTTAAGTTGCTTAAC-3′Cloned into expression strain E coli BL21 (DE3) and expressed proteinfor immunization experiments.

Construct 2:

Designed a primer to put YPT on the N-terminus of the PdB fusionconstruct containing TCE's and NEEK. RNYPT construct contains twocysteine linkers to ensure loop formation. Cloned into pET15b andexpressed for immunization experiments.

(SEQ ID NO: 30) 5′-CGCGCATATGGCTTGTAAAAAAGCCGAGGATCAAAAAGAAGAAGATCGCCGTAACTACCCAACCAATACTTACAAAACGCTTGAACTTGAATGTGCTGAGGGTGGTGCAAATAAAGCAGTAAATGAC-3′

Mouse Challenge for Protection:

Aim:

Compare immunogenicity and protective activity of PdB pneumolysoid, PdBlinear peptide fusion (construct 1) and PdB looped peptide fusion(construct 2).

Experimental Design:

Testing to determine immunogenicity and protective efficacy was done intwo parts.

Part 1: Immunogenicity of Toxoid Vs. Toxoid Fusion

Three groups of seven BALB/c mice (6 week old) were injected with 30 μgof PdB, PdB fusion linear peptide (PdB-2TCEs-linear NEEK), or PBS(saline). PHAD was used as adjuvant (200 μg/dose). Mice were boosted twoweeks after priming and again two weeks after first boost. Mice werebled 1 week after last boost and serum tested by ELISA at 1:450 dilutionfor anti-NEEK, anti-CbpA R2, and anti-PdB antibodies.

Results as shown in FIG. 3 demonstrate that immunization with the fusionprotein elicits antibodies against both PdB and CbpA while immunizationwith PdB alone does not elicit anti-CbpA antibodies.

Part 2: Protective Activity of Antibodies Generated by Toxoid Vs. TwoToxoid Fusions

Four groups of 10 BALB/c mice were primed subcutaneously with 10 μg ofimmunogen using Alhydrogel (3.2 mg/ml) as adjuvant and subsequentlyboosted twice at two week intervals. Mice were challenged the followingweek intranasally with 2.7×10⁷ S. pnuemoniae T4X and followed forsurvival.

Results shown in FIG. 4 demonstrate that the fusion with the looped YPTadduct was protective while the fusion with the linear NEEK adduct wasnot. This indicates that using the looped technology, a fusion proteincan be made that has the necessary tertiary structure to elicitprotective antibodies.

Conclusions from Example 1

Both linear and looped peptide PdB fusions are immunogenic for bothtoxoid and CbpA. However, the linear peptide PdB fusion does not elicitprotective antibody while the looped peptide PdB fusion is protective.This confirms fusions are immunogenic but the loop technology in neededin constructing fusions to toxoid to retain the native shape of thepeptide and therefore generate antibodies that are protective.

From this point forward for all examples only looped peptides of CbpAwere used.

Example 2: CbpA Peptide Fusions to Δ6N385 Pneumolysoid

Aim:

Δ6N385 is a pneumolysoid distinct from PdB of Example 1. This experimenttests if peptide fusion to one or both ends of Δ6N385 is protectiveagainst pneumococcal sepsis and meningitis in mice.

Construction of Fusions to Δ6N385 Pneumolysoid

Δ6N385 is a mutant form of pneumolysin that is non-toxic as disclosed inUS 2010/0166795, in particular paragraph 0068 and 0069 and in Mitchellet al., Molecular Microbiology 5:1883-1888, 1991. The following fusionproteins using Δ6N385 in pet33b as template were created:

1) Δ6N385-NEEK (SEQ ID NO: 20) (Δ6N385-KECAKEPRNEEKVKQCK)2) YPT-Δ6N385-NEEK (SEQ ID NO: 11)(ACKKAEDQKEEDRRNYPTNTYKTLELECAE-Δ6N385- KECAKEPRNEEKVKQCK)3) ΔV6N385-TCENEEK (SEQ ID NO: 21) (Δ6N385-qyikanskfigitqyikanskfigitggKECAKEPRNEEKVKQCK)* 4) YPT-Δ6N385 (SEQ ID NO: 22)(ACKKAEDQKEEDRRNYPTNTYKTLELECAE-Δ6N385) 5) YPT-Δ6N385-TCENEEK(SEQ ID NO: 23) (ACKKAEDQKEEDRRNYPTNTYKTLELECAE-Δ6N385-qyikanskfigitqyikanskfigitggKECAKEPRNEEKVKQCK) *lower case lettersdenote t-cell epitope (TCE)For construct Δ6N385-NEEK, primers PLYNdel(gcgcgcgccatatggcaaataaagcagtaaatgac) (SEQ ID NO: 31) and NEEKSac1(cgcgcggagctcctatttacattgcttaactttttcctcgtttcgaggttccttagcacactctttgtcattttctaccttatcctc)(SEQ ID NO: 32) were used to amplify by PCR. For constructYPT-Δ6N385-NEEK primers YPT(cgcgcatatggcttgtaaaaaagccgaggatcaaaaagaagaagatcgccgtaactacccaaccaatacttacaaaacgcttgaacttgaatgtgctgagggtggtgcaaataaagcagtaaatgac) (SEQ ID NO:33) and NEEKSacI were used. For construct Δ6N385-TCENEEK, primersJAT201b (cgcgtaacatatgatggcaaataaagcag) (SEQ ID NO: 34) and TCE-NEEK(2)(cgcggagctcctatttacattgcttaactttttcctcgtttcgaggttccttagcacactctttaccaccagtaataccaataaatttagaattagctttaatatattgaccaccagtaataccaataaatttagaattagctttaatatattgaccaccgtcattttctaccttatcctc)(SEQ ID NO: 35) were used. For construct YPT-Δ6N385, primers YPT andJAT215 (cgccgagctcctagtcattttctaccttatcctc) (SEQ ID NO: 36) were used.For construct YPT-Δ6N385-TCENEEK, primers YPT and TCE-NEEK(2) were used.For each construct, the PCR product was digested overnight with NdeI andSacI and ligated into prepared vector pet33b. Clones were sequenced bythe St. Jude Children's Research Hospital Hartwell Center. Clonescontaining the correct sequence were transformed into BL21(DE3)competent cells. Over night LB cultures were back diluted 1:50 intofresh media and shaken at 37° C. to OD₆₀₀=0.5. Cultures were inducedwith 0.07 mM IPTG overnight at 22° C. E. Coli was lysed using BugbusterHT reagent (Novagen) and purified over a Ni++ affinity column (Sigma).Protein was dialyzed into 10% glycerol/PBS. Endotoxin was removed andprotein was further diluted into 50% glycerol and stored at −20° C.

Mouse Challenge for Protection Against Two Different Strains ofPneumococci

Aim:

Compare unmodified Δ6N385 to peptide fusion on one or both ends ofΔ6N385 for immunogenicity and protective activity. Determine ifprotection is generated for both serotype 4 and serotype 2 pneumococci.

Experimental Design: This Experiment was Repeated in 3 Parts:

Part 1. Are Fusions to Both Ends of Δ6N385 Protective Against Serotype 4Meningitis and Death?

Mice used for this immunization were 6 week old female BalbC, 7 pergroup. Mice received 3 doses of antigen separated by 2 week intervals(Day 1, 15, 29). Mice were allowed to rest 3 weeks before challenge onday 50 with S. pneumoniae T4X. Bleeds for antibody titers (supernatantof a 75 μL bleed with heparanized capillary) were obtained byretro-orbital bleeding prior to immunization on day 1 and day 36. Foreach boost 10 μg of protein or 200 μg synthetic peptide were used with200 μg adjuvant PHAD. Antigens used were as follows:

1)  (SEQ ID NO: 8) Δ6N385 2)  (SEQ ID NO: 20) Δ6N385-NEEK 3) (SEQ ID NO: 22) YPT-Δ6N385 4)  (SEQ ID NO: 11) YPT-Δ6N385-NEEK 5) (SEQ ID NO: 21) Δ6N385-TCENEEK 6)  (SEQ ID NO: 23) YPT-Δ6N385-TCENEEK7)  PBS (-) control (adjuvant alone)

Antibody titers were determined by ELISA against plates coated overnightat 4 C with rCbpA or wild type pneumolysin (rPLY) (100 ng/well). Serumsamples were diluted 1/50, 1/150, 1/450 and 1/1050. Plates were blocked2 hours with 10% FBS and then incubated with diluted serum for one hourat room temperature. Plates were washed 5 times and incubated 1 hourwith anti-mouse IgG-AP (1:2000). Plates were washed 5× and incubated 20minutes in AP-yellow substrate (Sigma). OD₄₀₅ readings were taken. Thedata for 1:450 dilution is set forth in FIG. 5 which shows highanti-CbpA antibodies elicited by the N-terminal YPT and C-terminal NEEKlooped fusions.

Mice were challenged with T4X (1×10⁷ cfu) intratracheally. Meningitiswas determined by physical attributes (spinning of the head) andcollecting cerebrospinal fluid (CSF) for bacterial number. Survival wasmonitored daily for 2 weeks. The data for percent protection againstmeningitis is set forth in FIG. 6. The data for percent survival is setforth in FIG. 7. These data show that fusion to both ends ofpneumolysoid is protective against serotype 4 meningitis and death.

Part 2: Are Fusion Proteins Equal to or Better than Non-Fused Mixturesof Whole Proteins?

Mice used for this immunization were 6 week old female BalbC, 14 pergroup. Mice received 3 doses of antigen separated by 2 week intervals(Day 1, 15, 29). Mice were allowed to rest 3 weeks before intratrachealchallenge on day 50 with S. pneumoniae T4X. Bleeds for antibody titers(supernatant of a 75 μL bleed with heparanized capillary) were obtainedby retro-orbital bleeding prior to immunization on day 1 and day 36. Foreach boost 10 μg of protein was used with 100 μg adjuvant Alhydrogel(Sigma). Antigens used were as follows (fusions are #5 and 6):

1) WT PLY (SEQ ID NO: 5)

2) CbpA domain R2 (SEQ ID NO: 14)

3) Δ6N385 (SEQ ID NO: 8)

4) CbpA Domain R2 (SEQ ID NO: 14) mixed with Δ6N385 (SEQ ID NO: 8)

5) YPT-Δ6N385-NEEK (SEQ ID NO: 11)

6) YPT-Δ6N385-TCENEEK (SEQ ID NO: 23)

7) PBS (negative control, adjuvant alone)

Antibody titers were determined by ELISA against plates coated overnightat 4 C with rCbpA (100 ng/well). Serum samples were diluted 1/50, 1/150,1/450 and 1/1050. Plates were blocked 2 hours with 10% FBS and thenincubated with diluted serum for one hour at room temperature. Plateswere washed 5 times and incubated 1 hour with anti-mouse IgG-AP(1:2000). Plates were washed 5× and incubated 20 minutes in AP-yellowsubstrate (Sigma). OD₄₀₅ readings were taken. The data for the 1/450dilution is set forth in FIG. 8 and shows that the fusions areequivalent to mixtures of native proteins in generating antibody.

Mice were challenged with T4X (1×10⁷ cfu) intratracheally. Survival wasmonitored daily for 2 weeks. FIG. 9 provides the percent survival andshows that the fusion construct YPT-Δ6N385-NEEK is equivalent to themixture of Δ6N385 and CbpA R2 and both are superior to either nativeprotein alone.

Part 3: Are Mice Immunized with Fusion Protein of Sequence Serotype 4Protected if Challenged with a Strain of Pneumococci of Serotype 2?

Mouse Challenge

Mice used for this immunization were 6 week old female BalbC, 7 pergroup. Mice received 3 doses of antigen separated by 2 week intervals(Day 1, 15, 29). Mice were allowed to rest 3 weeks before intratrachealchallenge on day 50 with S. pneumoniae D39 (serotype 2). Bleeds forantibody titers (supernatant of a 75 μL bleed with heparanizedcapillary) were obtained by retro-orbital bleeding prior to immunizationon day 1 and day 36. For each boost 10 μg of protein was used with 100μg adjuvant Alhydrogel (Sigma). Antigens used were as follows (fusionsare #5 and 6):

1) WT PLY

2) CbpA domain R2 (SEQ ID NO: 14)

3) Δ6N385 (SEQ ID NO: 8)

4) CbpA Domain R2 (SEQ ID NO: 14) mixed with Δ6N385 (SEQ ID NO: 8)

5) YPT-Δ6N385-NEEK (SEQ ID NO: 11)

6) YPT-Δ6N385-TCENEEK (SEQ ID NO: 23)

7) PBS (negative control, adjuvant alone)

Antibody titers were determined by ELISA against plates coated overnightat 4 C with rCbpA (100 ng/well). Serum samples were diluted 1/50, 1/150,1/450 and 1/1050. Plates were blocked 2 hours with 10% FBS and thenincubated with diluted serum for one hour at room temperature. Plateswere washed 5 times and incubated 1 hour with anti-mouse IgG-AP(1:2000). Plates were washed 5x and incubated 20 minutes in AP-yellowsubstrate (Sigma). OD₄₀₅ readings were taken. Final antibody titers at1/450 dilution are shown in FIG. 10 and show the fusions are asimmunogenic as mixtures of native proteins (replicates FIG. 8 results).

Mice were challenged with D39X (3.5×10⁷ cfu) intratracheally. Survivalwas monitored daily for 2 weeks. FIG. 11 shows the percent survival andindicates the fusion is in the superior group of protection even ifchallenge is a heterologous serotype.

Conclusions from Example 2

Peptide fusion to Δ6N385 is immunogenic for both toxoid and CbpA.Peptide fusion to both ends of Δ6N385 is protective against meningitisand improves survival in an intratracheal challenge model. The fusionprotein vaccine provides protection against a strain of pneumococci of aserotype 2 that is different than the serotype 4 immunizing sequence andprotection is better than toxoid alone.

Significance:

This indicates that fusion of looped peptides and toxoid, specificallywith modifications at both ends of the toxoid protein, improves vaccineefficacy and is as good or better than mixtures of the native proteinsCbpA and pneumolysoid. Vaccine is effective across different serotypes.

Example 3: CbpA Peptide Fusions to L460D Pneumolysoid

Another toxoid of pneumolysin is the mutant changing the amino acid at460 from Leucine (L) to Aspartic acid (D) (called L460D). This isdisclosed in US 2009/0285846A1 by R. Tweten, herein incorporated byreference in its entirety. L460D is distinguished by more complete lossof the hemolytic activity of the native toxin than other pneumolysoidsPdB or Δ6N385. This would potentially make it a superior carrier forfused peptides as it might be a less toxic vaccine.

The experiments disclosed herein that make reference to L460D could havecontained either the pneumolysoid sequence set forth in SEQ ID NO: 7 orSEQ ID NO: 39. In all experiments, the two L460D pneumolysoids behavedidentically and will be referred to from this point forward as L460D.

Aim:

Compare L460D as a carrier for the CbpA peptides to other fusion/toxoidsfor immunogenicity and protective activity.

Construction of Fusions to L460D Toxoid

The following CbpA-L460D fusion proteins using L460D as template weregenerated.

1) YPT-L460D-NEEK (SEQ ID NO: 9) 2) YPT-L460D-TCENEEK (SEQ ID NO: 24)

For construct YPT-L460D-NEEK primers YPT (CGCGCATATGGCTTGTAAAAAAGCCGAGGATCAAAAAGAAGAAGATCGCCGTAACTACCCAACCAATACTTACAAAACGCTTGAACTTGAATGTGCTGAGGGTGGTGCAAATAAAGCAGTAA ATGAC) (SEQ ID NO:33) and NEEKSacI (cgcgcggagctcctatttacattgcttaactttttcctcgtttcgaggttccttagcacactattgtcattttctaccttatcctc) (SEQ ID NO: 32) wereused. For construct YPT-L460D-TCENEEK, primers YPT (SEQ ID NO: 33) andTCENEEK2(cgcggagctcctatttacattgcttaactttttcctcgtttcgaggttccttagcacactctttaccaccagtaataccaataaatttagaattagctttaatatattgaccaccagtaataccaataaatttagaattagctttaatatattgaccaccgtcattttctaccttatcctc) (SEQ ID NO: 35) were used.

For each construct, the PCR product was digested overnight with NdeI andSacI and ligated into prepared vector pet33b. Clones were sequenced bythe St. Jude Children's Research Hospital Hartwell Center. Clonescontaining the correct sequence were transformed into BL21(DE3)competent cells. Over night LB cultures were back diluted 1:50 intofresh media and shaken at 37° C. to OD600=0.5. Cultures were inducedwith 0.07 mM IPTG overnight at 22° C. E. Coli was lysed using BugbusterHT reagent (Novagen) and purified over a Ni++ affinity column (Sigma).Protein was dialyzed into 10% glycerol/PBS. Endotoxin was removed andprotein was further diluted into 50% glycerol and stored at −20° C.

Mouse Challenge: This Experiment was in Two Parts with DifferentChallenge BacteriaPart 1: Challenge with Serotype 4 Pneumococci

Mice used for this immunization were 6 week old female BalbC, 10 pergroup. Mice received 3 doses of antigen separated by 2 week intervals(Day 1, 15, 29). Mice were allowed to rest 3 weeks before challenge onday 50 with S. pneumoniae T4X. Bleeds for antibody titers (supernatantof a 75 μL bleed with heparanized capillary) were obtained byretro-orbital bleeding prior to immunization on day 1 and day 36.

For each boost 10 μg of protein was used with 100 μg adjuvant Alhydrogel(Sigma). Antigens used were as follows:

1) WT PLY (SEQ ID NO: 5)

2) Δ6N385 (SEQ ID NO: 8)

3) L460D (SEQ ID NO: 7)

4) YPT-L460D-NEEK (SEQ ID NO: 9)

5) YPT-L460D-TCENEEK (SEQ ID NO: 24)

6) PBS (−) (adjuvant alone)

Antibody titers were determined by ELISA against plates coated overnightat 4 C with rCbpA or rPLN (100 ng/well). Serum samples were diluted1/50, 1/150, 1/450 and 1/1050. Plates were blocked 2 hours with 10% FBSand then incubated with diluted serum for one hour at room temperature.Plates were washed 5 times and incubated 1 hour with anti-mouse IgG-AP(1:2000). Plates were washed 5× and incubated 20 minutes in AP-yellowsubstrate (Sigma). OD₄₀₅ readings were taken. The data showing theantibody titers at 1/450 dilution is shown in FIG. 12 and indicates thatfusions to L460D show superior antigenicity.

Anti-sera against L460D, YPT-L460D-NEEK and PBS (−) control were alsotested for functional activity for binding to S. pneumoniae T4R wholebacteria in an ELISA based assay. Plates were coated with 1×10⁶ cfu/welland ELISA protocol as previously described was used. The data is shownin FIG. 13. Antibody binding to intact bacteria was highest in the groupYPT-L460D-NEEK.

Mouse Challenge:

Mice were challenged with T4X (1×10⁷ cfu) intratracheally. Survival wasmonitored daily for 2 weeks. FIG. 14 shows the percent survival.

Fusion of L460D with two peptides is superior to one peptide and L460Dis a superior carrier than Δ6N385.

Part 2: Challenge with Serotype 2 Pneumococci

Mouse Challenge

Mice used for this immunization were 6 week old female BalbC, 10 pergroup. Mice received 3 doses of antigen separated by 2 week intervals(Day 1, 15, 29). Mice were allowed to rest 3 weeks before challenge onday 50 with S. pneumoniae D39. Bleeds for antibody titers (supernatantof a 75 μL bleed with heparanized capillary) were obtained byretro-orbital bleeding prior to immunization on day 1 and day 36.

For each boost 10 μg of protein was used with 100 μg adjuvant Alhydrogel(Sigma). Antigens used were as follows:

1) WT PLY (SEQ ID NO: 5)

2) Δ6N385 (SEQ ID NO: 8)

3) L460D (SEQ ID NO: 7)

4) YPT-L460D-NEEK (SEQ ID NO: 9)

5) YPT-L460D-TCENEEK (SEQ ID NO: 24)

6) PBS (−) (adjuvant alone)

Antibody titers were determined by ELISA against plates coated overnightat 4 C with rCbpA and rPLN (100 ng/well). Serum samples were diluted1/50, 1/150, 1/450 and 1/1050. Plates were blocked 2 hours with 10% FBSand then incubated with diluted serum for one hour at room temperature.Plates were washed 5 times and incubated 1 hour with anti-mouse IgG-AP(1:2000). Plates were washed 5× and incubated 20 minutes in AP-yellowsubstrate (Sigma). OD₄₀₅ readings were taken. The data for 1/450dilution is shown in FIG. 15 and indicates that the fusion constructelicits the highest antibody for both CbpA and Pln.

Mice were challenged with D39X (1×10⁷ cfu) intratracheally. Survival wasmonitored daily for 2 weeks. The percent survival is shown in FIG. 16.

The fusion of two peptides to L460D is protective against serotype 2strain D39 which is a different serotype than the sequences of theimmunogen. This establishes cross-serotype protection.

Conclusions from Example 3

L460D is superior to Δ6N385 as a carrier for CbpA fusion peptides interms of immunogenicity and generation of protective antibody. Crossserotype protection was demonstrated.

Example 4. Protection from Neisseria Meningitis

Neisseria meningitidis is an important cause of meningitis. Neisseriaand pneumococcus share binding to the same laminin receptor protein atthe blood brain barrier to initiate meningitis. This property is carriedby the NEEK portion of CbpA and Neisseria carry a protein cross reactivewith NEEK. Thus, a vaccine containing NEEK might also react withNeisseria and prevent meningitis.

Aim:

Determine if peptide toxoid fusion confers cross protection againstNeisseria meningitis.

Fusion Confers Protection from Neisseria Meningitis

Mice used for this immunization were 6 week old female BalbC, 8 pergroup. Mice received 3 doses of antigen intraperitoneally separated by 2week intervals (Day 1, 15, 29). For each boost 10 μg of protein was usedwith 100 μg adjuvant Alhydrogel (Sigma). Antigens used were as follows:

1) L460D (SEQ ID NO: 7)

2) YPT-L460D-NEEK (SEQ ID NO: 9)

3) CbpA R2 domain (SEQ ID NO: 14)

4) PBS (negative control, adjuvant alone)

Mice were challenged intraperitoneally on day 50 with 1×10⁶ cfuNeisseria meningitidis group a. Survival and presence of meningitis wasmonitored for 1 week. FIG. 17 shows the percent survival and indicatesthe fusion was protective against death by Neisseria. FIG. 18 shows thatthe fusion protein is protective against meningitis compared to toxoidalone or negative control.

Conclusion from Example 4

Peptide toxoid fusion confers significant protection across species frommeningitis and death due to either pneumococcus or meningococcus.

Example 5: Protection by the Fusion Protein is Due to Both CbpA andToxoid Components

Aim:

Dissect protective activity to document that peptide and toxoid eachcontribute to protection by the fusion construct.

Two isogenic mutants were used as challenge strains to dissectprotective activity. D39X expresses both toxoid and CbpA; CbpA-D39X hasa deletion of CbpA. It would be expected that a vaccine would need toelicit antibodies to both toxin and CbpA to counter an infection withD39X. It would be expected that a vaccine would need to elicitantibodies only to toxin to counter an infection by a bacteria notexpressing CbpA.

Mice used for this immunization were 6 week old female BalbC, 30 pergroup. Mice received 3 doses of antigen separated by 2 week intervals(Day 1, 15, 29). Bleeds for antibody titers (supernatant of a 75 μLbleed with heparanized capillary) were obtained by retro-orbitalbleeding prior to immunization on day 1 and day 36.

For each boost 10 μg of protein was used with 100 μg adjuvant Alhydrogel(Sigma). Antigens used were as follows:

1) L460D (SEQ ID NO: 7)

2) YPT-L460D-NEEK (SEQ ID NO: 9)

3) PBS (−) (adjuvant alone)

Mice were challenged on day 50 with 1×10⁷ cfu of either D39X or anisogenic mutant CbpA−/D39X (10 mice from each immunized group used perstrain). No differences were seen in the course or titer of the twostrains of bacteria in blood. Survival was monitored daily for 2 weeks.As shown in FIG. 19, fusion toxoid showed enhanced survival protectionover L460D alone when both pneumolysin and CbpA were on the bacteria(i.e. D39X). Fusion was not better than L460D alone if there was no CbpAon the bacteria (CbpA-D39X). These differences indicate that the toxoidand the CbpA peptide components of the fusion both contribute toprotection.

Conclusion from Example 5

Both parts of the fusion construct, the peptides and the carrier,contribute to the protective activity of the vaccine when wild typebacteria expressing both CbpA and Pln are the challenge.

Example 6: Efficacy of Peptide-Toxoid Fusion in Colonization Model

Pneumococci initiate infection by colonizing the nasopharynx beforespreading to the lung or blood. A vaccine that could preventcolonization would be desirable. The YPT portion of the fusion vaccineis designed to block translocation of bacteria from the nasopharynx tothe lung/blood. For vaccines to have activity on a mucosal surface suchas the nasopharynx, they typically need to be administered to the mucosadirectly, i.e. intranasally. A vaccine that could elicit protection fromcolonization even when given parenterally would be desirable.

Aim:

Determine if immunization with peptide-toxoid fusion intraperitoneally(IP) decreases colonization of the nasopharynx.

Fusion Active Against Colonization (Data from Two Experiments)

Mice used for this immunization were 6 week old female BalbC, 7 pergroup. Mice received 3 doses of antigen intraperitoneally separated by 2week intervals (Day 1, 15, 29). For each boost 10 μg of protein was usedwith 100 μg adjuvant Alhydrogel (Sigma). Antigens used were as follows:

1) L460D (SEQ ID NO: 7)

2) YPT-L460D-NEEK (SEQ ID NO: 9)

3) CbpA R2 (SEQ ID NO: 14)

4) PBS (−) (adjuvant alone)

Mice were infected intranasally on day 50 with 1.5×10⁷ cfu T4X. Nasallavages were taken at 24, 48, 72 and 96 hours and plated for bacterialnumbers to indicate extent of colonization. Results are shown in Table2.

TABLE 2 Log CFU in nasopharyngeal wash following IP vaccine YPT-L460D-L460D NEEK CbpA R2 PBS(−) Exp 1 48 h 5.8 6.5 6.4 6.0 96 h 5.6 4.9*4.8* >8 Exp 2 48 h 5.7 6.3 6.2 6.0 96 h 5.7 5.2* 5.1 >8 *Significantdifference between 48 h and 96 h

Conclude:

Negative control animals (PBS) experienced a 2.5 log increase inbacterial titer in nasopharynx between 48 to 96 h. L460D animals stayedthe same as original inoculum. CbpA R2 (positive control) andYPT-L460D-NEEK decreased nasopharyngeal bacterial numbers by 1.5 logsand were 3 logs less than PBS control at 96 h.

Conclusion for Example 6

IP immunization with peptide-toxoid fusion prevents growth of bacteriain model of colonization of the nasopharynx.

Significance:

Activity in controlling colonization of the nasopharynx is detectablefor YPT-L460D-NEEK even with immunization IP.

Example 7: Does Protection in the Nasopharynx Arise from IL-17 T Cellsand/or B Cells in Nasal Associated Lymphoid Tissue (NALT) PostImmunization

Protection from systemic diseases is believed to be due to antibodymediated defenses. On mucosal surfaces such as the nasopharynx, T cellsand B cells cooperate. In particular IL-17 is believed to be importantin recruiting cells to the nasopharynx to eliminate colonization. Tomeasure T and B cells in the nose, the NALT must be harvested and cellsseparated to enumerate and determine specific cell markers foridentification. In our model, NALT contains ˜2% T cells producing IL-17in naïve colonized mice and IL-17 cytokine production peaks at day 10post immunization.

Method to Quantitate NALT T Cells Post Vaccine:

Groups of 5 mice were immunized either intranasally or intraperitoneally3 times in two week intervals with L460D, YPT-L460D-NEEK fusion, or CbpAR12. Adjuvant was cholera toxin (CT, not heat inactivated). For the CTalone group, NALT was harvested before final boost for baseline. Forremaining groups, NALT and spleen were harvested 10 days after the finalboost and assayed by

Harvest of NALT yielded ˜5×10⁵ T cells, a value in range of previouscontrols in naïve and colonized mice. Spleens yielded 1×10⁷ cells.100,000 cells were counted and tested by:

1) FACS for pneumcoccal-reactive T cells bearing CD4⁺, CD19⁺ or IL-17⁺

2) ELISPOT for B cells making antibody to CbpA or Pln

T Cell Characteristics from NALT and Spleen

Results are shown in Table 3 and show that immunization IN with CbpA orthe fusion invoked a small increase in IL-17 T cells compared to CTalone only in NALT. This was greater than if immunization was IP.

TABLE 3 Characteristics of T cells elicited by fusion vaccine % TCells + for IL-17 NALT Spleen CbpA IN 3.8 0.2 L460D IN 2.1 0.2 Fusion IN3.3 0.2 CT alone IN 2.9 0.2 CbpA IP 0.6 0.2 L460D IP 2.1 0.3 Fusion IP2.6 0.2 CT alone IP 2.3 0.2

B Cell Results:

ELISPOT:

1×10⁵ B cells were seeded per well of a 96 well plate. Cells wereincubated with antigen (CbpA or L460D) and antibody production inresponse to stimulation by specific antigen was indicated by developmentof colored spots. Maximum is ˜40 spots/filter. The data (number ofpositive spots per well reactive to CbpA or L460D) is set forth in FIG.20 as a function of 4 immunizing antigens, given by 2 routes.

B cells in NALT were strongest with intranasal route of immunization.However, it is notable that reactive B cells were detected even afterintraperitoneal immunization with L460D and Fusion. Fusion inducedstrong response to CbpA as expected but also strong to pneumolysoid.

Conclusion from Example 7

The fusion construct elicits a small increase in IL-17⁺ T cells and astrong increase in B cells reactive for anti-CbpA and anti-pneumolyin inthe NALT. More activity was seen with IN than IP administration.

Example 8: CbpA-Pneumolysin Fusion Protein Expressed/Purified fromTagless Vector

For vaccines used in humans, constructs that do not express a tag suchas poly-histidine are desired.

YPT-L460D-NEEK was amplified from the pet33 construct using oligosYPTNDE (cgcgcgcgcatatggcttgtaaaaaagccgagg) (SEQ ID NO: 37) and NEEKSAC(SEQ ID NO: 38). The PCR product was cut overnight with NdeI and SacIand ligated into prepared tagless vector pET27b. Clones were sequencedby the St. Jude Children's Research Hospital Hartwell Center. Clonescontaining the correct sequence were transformed into BL21(DE3)competent cells. Protein expression/purification was carried out by StJude Children's Research Hospital's Protein Production Facility.

Test Constructs Using Tagless CbpA Pneumolysin Fusion

Mice used for this immunization were 6 week old female BalbC, at least40 per group in total, (i.e. 10 per group) in multiple experiments. Micereceived 3 doses of antigen separated by 2 week intervals (Day 1, 15,29). Bleeds for antibody titers (supernatant of a 75 μL bleed withheparanized capillary) were obtained by retro-orbital bleeding prior toimmunization on day 1 and day 36.

For each boost 10 μg of protein was used with 100 μg adjuvant Alhydrogel(Sigma). Antigens used were as follows:

1) YPT-L460D-NEEK (SEQ ID NO: 9)

2) L460D-NEEK (SEQ ID NO: 25)

3) PBS (−) (adjuvant alone)

Mice were challenged on day 50 with 1×10⁷ cfu T4X intratracheally.Meningitis was determined by physical attributes (spinning of the head)and collection of cerebrospinal fluid (CSF) for bacterial number.Survival was monitored daily for 2 weeks. The data is summarized in FIG.21 and then shown for each mouse in FIGS. 22 to 25.

As shown in FIG. 22, mice immunized with YPT-L460D-NEEK had asignificantly lower incidence of meningitis than mice immunized withL460D toxoid or adjuvant alone. As shown in FIG. 23, mice immunized withYPT-L460D-NEEK or L460D toxoid were protected similarly and both weresignificantly better than adjuvant alone.

As shown in FIG. 24, mice immunized with YPT-L460D-NEEK had asignificantly lower incidence of meningitis than mice immunized withL460D toxoid or adjuvant alone.

As shown in FIG. 25, L460D and YPT-L460D-NEEK show excellent protectionfollowed by L460D-NEEK.

Conclusion from Example 8

YPT-L460D-NEEK and L460-NEEK were significantly better than L460D alonein preventing meningitis and death.

Example 9: Test Pneumolysoids Δ6N385 and L460D and their Fusions forResidual Hemolytic Activity and Determine if they Elicit AntibodyNeutralizing Toxin-Induced Hemolysis

For use in humans, the pneumolysoids must be nontoxic and generateantibody that can neutralize the hemolytic (cytotoxic) activity of thewild type toxin. The fusion must not increase toxic activity.

Aim:

Quantify residual toxic activity of toxoids and whether fusion altersthis property. Determine relative ability of toxoids±fusions to elicitantibody capable of neutralizing toxic activity of native Pneumolysin.

Toxoid Constructs Remained Nonhemolytic

Recombinant proteins were tested for hemolytic activity against sheepred blood cells. Proteins were diluted to 10 μg/ml concentration in PBS.Protein was then serial diluted 1:2 with a starting concentration of 1μg/ml in dilution buffer (10 ml PBS, 0.01 g BSA, 0.015 g DTT) in a 96well V-bottom plate. 50 μL from each well was transferred to a freshV-bottom plate. 1 ml of defibrinated sheep blood was washed 3× in 10 mlPBS (blood was centrifuged 5 minutes at 1000×g). 50 μL blood was addedto each well containing protein and the plate was incubated 30 minutesat 37′C. The plate was centrifuged 3 minutes at 1000×g and supernatantwas transferred to a 96 well ELISA plate. OD540 readings were taken witha plate reader. 1% Triton X-100 was used as a positive control withdilution buffer as the negative control. The experiment was repeatedtwice and the results combined and shown in FIG. 26. Wild typepneumolysin is hemolytic and this activity titrates out with dilution.L460D and Δ6N385 are non-hemolytic even at high concentrations. Fusionsshow same profile as the pneumolysoid on which they are attached.

Pneumolysin Neutralization Assays Using Antisera from Immunized Mice

Scrum samples from immunized mice were tested for anti-hemolyticactivity with wild type pneumolysin. Briefly, 1.2 μL scrum was mixedwith 6 μL 10% glycerol and 52.8 μL PBS 1 hour at room temperature toremove inhibitory cholesterol. Samples were spun 5 minutes at 10,000×gand 50 μL supernatant was transferred to a V-bottom plate. Samples wereheat inactivated at 56° C. for 30 minutes to remove complement. Make2-fold serial dilutions of sera in PBS and add 50 μL wild typepneumolysin (4 hemolytic units). Incubate 15 minutes at 37° C. Prepare1% rabbit blood by washing 1 ml blood in 10 mls PBS 3 times. Add 0.001%beta-mercaptoethanol to final volume of blood. Add 50 μL to each welland incubate 30 minutes at 37° C. Spin plates 3 minutes at 1000×g andtransfer 60-80 μL to fresh ELISA plate. Read absorbance at 540 nm.Anti-PdB antiserum was used as the positive control and results areshown in FIG. 27. Titer of antibody neutralizing wild type toxinhemolytic activity was greatest after immunizing with YPT-L460D-NEEKmore so than L460D which was more so than Δ6N385.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “all element” means one or more element.

TABLE 4 SEQ ID NO: AA/NT Description 1 AA R2₁ fragment sequence of CbpAof S. pneumoniae 2 AA R2₂ fragment sequence of CbpA of S. pneumoniae 3AA Cysteine mutant of R2₁ fragment sequence of S. pneumoniae (AKA “YPT”;looped) 4 AA Cysteine mutant of R2₂ fragment sequence of S. pneumoniae(AKA “NEEK”; looped) 5 AA Wild-type pneumolysin amino acid sequence ofS. pneumoniae (AKA “PLY”) 6 NT Wild-type pneumolysin nucleotide sequenceof S. pneumoniae 7 AA L460D pneumolysoid sequence 8 AA Δ6N385pneumolysoid sequence 9 AA YPT-L460D-NEEK fusion protein sequence 10 NTYPT-L460D-NEEK fusion protein nucleotide sequence 11 AA YPT-Δ6N385-NEEKfusion protein sequence 12 AA Full-length CbpA amino acid sequence of S.pneumoniae 13 AA CbpA R1R2 amino acid sequence of S. pneumoniae 14 AA R2domain amino acid sequence of CbpA of S. pneumoniae 15 AA TCE1 sequence16 AA TCE2 sequence 17 AA PdB pneumolysoid amino acid sequence 18 AAPdB-2TCEs-linear NEEK fusion protein sequence 19 AA Looped YPT-PdB-2TCEs-linear NEEK fusion protein sequence 20 AA Δ6N385-NEEK fusionprotein sequence 21 AA Δ6N385-TCE-NEEK fusion protein sequence 22 AAYPT-Δ6N385 fusion protein sequence 23 AA YPT-Δ6N385-TCE-NEEK fusionprotein sequence 24 AA YPT-L460D-TCE-NEEK fusion protein sequence 25 AAL460D-NEEK fusion protein sequence 26 NT JAT201 primer 27 NT C-termFusion primer 28 NT JAT209 primer 29 NT JAT210 primer 30 NT Construct 2primer 31 NT PLYNde1 primer 32 NT NEEK Sac1 primer 33 NT YPT primer 34NT JAT201b primer 35 NT TCENEEK2 primer 36 NT JAT215 primer 37 NT YPTNDEprimer 38 NT NeekSac primer 39 AA L460D pnemolysoid variant sequence

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A fusion protein comprising a) a first polypeptide having at least70% sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQID NO: 2, wherein said first polypeptide forms a loop conformation andis immunogenic, b) a second polypeptide, fused in frame to said firstpolypeptide, said second polypeptide comprises at least one T cellepitope (TCE), wherein said TCE is heterologous to said firstpolypeptide, and c) a third polypeptide, fused in frame to said first orsaid second polypeptide, wherein said third polypeptide is from abacteria and is immunogenic. 2-34. (canceled)